Molecular Vision 2003; 9:31-42 <http://www.molvis.org/molvis/v9/a6>Received 24 October 2002 | Accepted 8 February 2003 | Published 15 February 2003

The neural retina in vertebrates exhibits a remarkablelaminar architecture of alternate cellular and synaptic layers,and a striking pattern of mosaic organization where neuronsof the same type lying in each corresponding cellular layerare distributed in a non-random array intermingled with otherneuronal cells [1-4]. The mosaic organization of photorecep-tor cells in most vertebrate retinas has been used as a modelsystem for the study of neural patterning and organization [4-7], and it is believed to be a developmental strategy of thecentral nervous system for facilitating connections of the vi-sual neural circuitry and efficient sampling and processing ofvisual signals [1,8,9]. Although the cone photoreceptor mo-saic has been examined in a variety of species [1,2,6,10-14],few studies have described the development of the mosaicpattern quantitatively, and little is known about the mouse dueto the lack of adequate developmental markers. A quantitative

study of the spatial organization and mosaic development ofcone photoreceptors in the mouse retina is particularly impor-tant because there is an increasing number of spontaneous andinduced mouse mutants that offer unique views of the retinalstructure, function and disease [15-22]. Understanding thecellular organization of the mouse retina and the normal de-velopmental process of the mouse cone mosaic will be criticalfor studying the pathological processes in mutant mouse mod-els of human retinal degeneration.

Investigating the development of mouse cone mosaic re-quires early appearing, cell specific markers to identify thedeveloping cone cells. However, currently used markers forthe mouse cones [23-26] appear to be inadequate for this task.In a previous study, transgenic mice with the living cone cellslabeled by a transgenic green fluorescent protein (GFP) markerwere created [27]. This transgenic cone marker was capableof selectively labeling the entire cone cell beginning from theearly developmental stage. The goal of the present study wasusing the transgenic mice to study the development and pat-terning of cone photoreceptors in the mouse retina.

© 2003 Molecular Vision

Development of the cone photoreceptor mosaic in the mouse retinarevealed by fluorescent cones in transgenic mice

Yijian Fei

Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, CT

Purpose: Normal function of the retina relies on the orderly stereotyped organization of different neurons and theirsynaptic connections. How such neural organization is patterned during development remains poorly understood due tothe paucity of adequate developmental markers. This study was to examine the spatial organization and development ofcone photoreceptors quantitatively in the mouse retina.Methods: A transgenic approach was used to generate a living cone cell marker by driving GFP expression in mousecones with the human red/green opsin gene 5' sequences. The spatial organization and development of the cones in themouse retinas were examined quantitatively with epifluorescence and scanning laser confocal microscopy. Cone specificGFP expression in the developing retinas was verified with peanut agglutinin (PNA) staining. Developmental expressionof mouse cone opsin genes was determined with RT-PCR.Results: The fluorescent retinal cells expressing GFP can be visualized as early as on embryonic day E15. Following upmorphological differentiation of these cells revealed features that were consistent with the typical morphology of themouse cones. Double labeling with cone specific PNA showed that these cells were co-labeled starting from postnatal dayP1, and that a subpopulation of PNA positive cones expressed the GFP. The fluorescent cell densities had a similar ventraland dorsal distribution from E15 to P2, increased dramatically in the ventral by P6, and in the dorsal from P7. Nearestneighbor distance analysis demonstrated that this subpopulation of cones was organized into a regular mosaic pattern witha regularity index of 4.82 in the central and 3.55 in the peripheral retina. Quantitative pattern assessment of the developingcones revealed that the fluorescent cells appeared to be distributed in a non-random array before birth. The regularity ofthe cone array began to rise on P7, in parallel with the onset of mouse green opsin gene expression and the developmentof cone pedicles. The regular pattern of cone mosaic organization was basically formed by P10, coinciding with the timingof the cone pedicle maturation.Conclusions: The cones in the mouse retina are organized in a regular mosaic pattern. Patterning the cone mosaic appearsto follow a two phase developmental process involving regulated opsin gene expression and cone pedicle maturation: anearly phase where a non-random array emerges during cone differentiation, and a late phase where the regular mosaicpattern is mature at the time when cone synaptic contacts are being formed.

Correspondence to: Yijian Fei, MD, Department of Internal Medi-cine, Yale University School of Medicine, LMP 2073, PO Box208029, New Haven, CT, 06520; Phone: (203) 785-6044; FAX: (203)785-7068; email: yijian.fei@yale.edu

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© 2003 Molecular VisionMolecular Vision 2003; 9:31-42 <http://www.molvis.org/molvis/v9/a6>

METHODSGeneration and use of the transgenic mice: Procedures forcreating the transgenic mice with GFP expression in the coneswere described elsewhere [27]. The transgene was composedof a 6.8 kb regulatory sequence 5' of the human red and greenopsin genes containing the promoter and locus control regionisolated from the pR6.5 lacZ plasmid [24] (a generous giftfrom Dr. Jeremy Nathans, Johns Hopkins University,) and theGFP reporter cassette. Transgenic mice were identified bypolymerase chain reaction (PCR) analysis of mouse tail DNA.Mice used in this study did not carry the rd allele from thebackground SJL strain. Prenatal mice were from timed preg-nancies of the transgenic mice. The morning when the copu-lation plug was observed was taken as day 0. Mice used inthis study were cared for and handled in accordance with theYale Animal Care and Use Committee guidelines and theARVO Statement for the Use of Animals in Ophthalmic andVision Research.

Preparation of retinal wholemounts and sections: Miceused in this study were killed by inhalation of overdose ofanesthetic, Isofluorane (Fort Dodge Animal Health, FortDodge, Iowa) or Methoxyflurane (Pitman-Moore Inc.,Mundebin, IL). The orientation of mouse eyes was markedthrough lightly burning the cornea at 12 o’clock position witha fine tipped surgical cautery before the eyes were enucleated.The neural retinas were dissected free in ice cold Hanks’ buffer(Ca2+ and Mg2+ free) under a dissection microscope. For ex-amination of the live cone cells, some retinas were freshlydissected from the enucleated eyes without fixation. For bet-ter preservation of the retinal architecture, other retinas werefixed before dissection by immersing the enucleated eyes inice cold 4% paraformaldehyde in phosphate buffered saline(PBS, pH 7.4). The time of fixation was 30-60 min for theprenatal mouse eyes, and overnight for the postnatal eyes. Thefixed mouse eyes were rinsed with PBS 3 times, 30-40 mineach. The dissected neural retinas were flat mounted on glassslides with the photoreceptor side up, coverslipped with ei-ther PBS (for live retinas) or 90% glycerol in PBS (for fixedretinas). For sections, the fixed retinas from adult transgenicmice were embedded in 5% agarose. Cross sections (50-200µm) were cut with a vibratome (Electon Microscopy Sciences,Fort Washington, PA). The nuclei in some retinal sections werecounterstained with propidium iodide (2 µg/ml) for about 1 h,and then rinsed in PBS for 30-60 min.

Double labeling of retinal wholemounts with peanut ag-glutinin (PNA): To determine the specificity of GFP labelingof the cones during development, cone cell specific PNA [23]was used to stain the retinal wholemounts. The fixed retinaswere incubated in PBS containing 10% normal goat serumand 0.2% Triton X-100 for 1-3 h, rinsed with PBS, and re-acted with 1:10 diluted rhodamine-conjugated PNA (VectorLaboratories, Burlingame, CA) overnight. PNA stained reti-nas were rinsed with PBS, and mounted on glass slides with80% glycerol in PBS containing 0.4% phenylendiamine.

Epifluorescence and confocal microscopy: To detect theGFP signal from mouse cone cells expressing the GFP

transgene, retinal preparations were first examined with a Zeissmicroscope equipped with a Micromax CCD camera(Princeton Instruments, Trenton, NJ) and standard HQ FITCfilter sets (Chroma, Brattleboro, VT). Some images of the reti-nal wholemounts were also taken with differential interfer-ence contrast (DIC) optics to view the overall profiles of boththe fluorescent and non-fluorescent retinal cells. A Biorad MRC600 scanning laser confocal microscope (Hercules, CA) fittedwith standard FITC and rhodamine filter sets was used to col-lect 3 dimensional images from the retinal preparations. Thedigital images were analyzed with IPLabs software(Scanalytics Inc., Fairfax, VA). Montages were assembled fromoverlapping images taken from the retinal wholemounts basedon common reference points. IMARIS software (Bitplane AG,Zürich, Switzerland) was used to process the confocal 3D datasets on a SGI silicon graphics octane 2 work station (SiliconGraphics, Inc., Mountain View, CA) for 3D view of the conecell morphology. Image manipulations involved adjusting con-trast, adding scale bars and labels. None of the values in theimage files were manipulated. The final figures were com-posed with Adobe Photoshop 5.5.

Sampling and quantitative analysis of the cone mosaic:Transgenic mice used for quantitative analysis of cone mo-saic were chosen from line 5933, since this line had the high-est level of GFP expression in the mouse cones [27]. Threeretinas of three mice were analyzed at each age (E19, P2, P6,P7, P9, P10 and adult). To examine the topographic distribu-tion of the fluorescent cones during development, the num-bers of the fluorescent cones were counted in contiguous sam-pling windows (300 µm x 132 µm/window for adult and 100µm x 132 µm/window for developing mice) from the dorsalperiphery through the optic disc to the ventral periphery. Thecone density data were analyzed with IGOR Pro software(WaveMetrics, Lake Oswego, OR). Spatial density profiles ofthe fluorescent cones were plotted as the average cone density(cones/mm2) against the retinal eccentricity (mm). The near-est neighbor distance analysis [1,7,28-32] was used to quan-tify the regularity of the cone mosaic pattern. Cone nearestneighbor distances were measured from center to center ofthe nearest cone cell bodies using IPLabs software. Fields forthis analysis were chosen from the central area of the dorsalretina, where the fluorescent cones had the highest densitysimilar to that in non-transgenic mouse [33] and GFP express-ing cones approached 100% of the total cones revealed bycounterstaining with PNA [27]. The far periphery of the dor-sal retina where the cell density is quite low was also sampledin adult retinas to examine whether the regularity of conemosaic pattern in the central retina would be different fromthat in the peripheral retina. The distribution of the nearestneighbor distances was analyzed with IGOR Pro software(WaveMetrics, Lake Oswego, OR) and fitted to the normalGaussian distribution. The statistical difference between thesample distribution and the normal distribution was determinedwith the χ2 test [1]. The regularity index (RI) or conformityratio [29], was calculated as the ratio of the mean nearest neigh-bor distance to the standard deviation, which provides a goodtest for mosaic regularity [1,28,29,32].

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RNA preparation and reverse transcription-PCR: TotalRNA was isolated from mouse retinas from E13 to P10 withTRIzol reagent (Life Technologies, Gaithersburg, MD) fol-lowing the manufacturer’s instructions. Aliquots of the RNAsamples with or without DNase I (RNase free) treatment wereused for RT-PCR. The mouse retinal mRNA was reversetranscripted into cDNA using the ThermoScript RT-PCR sys-tem (Life Technologies, Gaithersburg, MD) with the Oligo

[dT]20

primer, and amplified by PCR according to themanufacturer’s protocols. PCR primers used to amplify themouse blue and green cone opsin (short and middle wave-length sensitive opsin, respectively) gene cDNA [34,35] andthe PCR cycling profiles were described in the previous study[27].

RESULTSCone specific, consistent pattern of GFP transgene expres-sion: It was previously shown that the GFP expression reli-ably marked the cones in the adult mouse retina [27]. Exami-

© 2003 Molecular VisionMolecular Vision 2003; 9:31-42 <http://www.molvis.org/molvis/v9/a6>

Figure 1. Fluorescent cells are restricted to the photoreceptor celllayer. These are projected confocal images from 36 frames of apropidium iodide counterstained cross retinal section from an adulttransgenic mouse taken with standard FITC/Rhodamine filter setsand a 20x lens. A: Image (pseudocolored green) of the GFP express-ing fluorescent cells. B: Rhodamine image (pseudocolored red) ofthe same filed, showing the three nuclear layers and the two synapticlayers of the mouse neural retina. C: The merged image, showing thelocation of the majority of fluorescent cell bodies in the outer borderof the outer nuclear layer (ONL) and the cone pedicles (CP) in theinner part of the outer plexiform layer (OPL). No cells in other reti-nal layers exhibit GFP fluorescence. CS: outer and inner segments ofphotoreceptor cells. CB: cell bodies. CE: axons. INL: inner nuclearlayer. IPL: inner plexiform layer. GC: ganglion cell layer. Scale barrepresents 50 µm.

Figure 2. Fluorescent photoreceptor cells are cones. These are con-focal images of a representative fluorescent cell taken from a PNAlabeled flat mounted retina. A: 3D image showing the morphologicalfeatures of mouse cones including a conical shaped outer segment(OS), a thick inner segment (IS) and a large synaptic pedicle (P)connected to the cell body (C) via an axon (A). B: Co-labeling of thisfluorescent cone (green) by cone specific PNA marker (red) in theouter segment (OS). Note that these images were from the mid-pe-ripheral ventral retina. The cone cell does not appear to stand up-right, which is likely caused by the coverslipping process that mightpush the cells to bow forward. There are 2-3 PNA+ cells in B that didnot express GFP, which is common in the ventral retina. Scale barrepresents 10 µm.

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nation of retinal wholemounts and sections further demon-strated that the GFP labeling was restricted to the photorecep-tors and that no other retinal cells expressed detectable levelsof GFP (Figure 1). These fluorescent photoreceptor cells hadthe majority of their somata located in the outer border of theouter nuclear layer (ONL) and their pedicles lying in the innerpart of the outer plexiform layer (OPL; Figure 1C). A 3D im-age of one of the fluorescent cells from a flat mounted retinarevealed a conical shaped outer segment, a thick inner seg-ment and a large flattened synaptic pedicle connected to thecell body via an axon (Figure 2A). All these features are con-sistent with the morphology of a cone cell in the mouse retina[36]. Double labeling with cone specific PNA marker showedco-localization of the staining in the outer segment (Figure2B), which further confirms that the fluorescent photorecep-tor cells are indeed the cones. All three transgenic lines gener-ated had a similar dorsal-ventral graded pattern of GFP ex-pression despite of a great variation in the number of conesexpressing GFP. This characteristic expression pattern has beenfaithfully transmitted from early generation (F1) to later ones(F4). Figure 3 shows the representative patterns of fluores-cent cones labeled by GFP in the F1 (Figure 3A,B) and F4(Figure 3C,D) mouse retinas of the same 5933 line. Both miceexhibited very similar spatial patterns of the fluorescent conedistributions in the dorsal (A and C) and the ventral (B and D)retinas. The mosaic organization of cones in the mouse retinais readily appreciated in Figure 3, where the fluorescent conesappeared to be somewhat evenly spaced.

Early emergence and morphological differentiation of thefluorescent cells: To determine when the fluorescent cells

could be detected during retinal development, retinalwholemounts from the transgenic lines at each embryonic daystarting from E13 when cone genesis begins [37] were exam-ined. The first fluorescent cells that could be reliably detectedin the mouse retinas from line 5933 appeared at embryonicday E15 in both live (Figure 4) and fixed retinas. These fluo-rescent cells were situated in the outer surface of the neuro-blast layer of the embryonic retina, and most of them had asclerally oriented process with a bulbous enlargement at theend. Similar fluorescent cells were also observed in the E15retinas of another mouse line 5922 (Figure 5), where the fluo-rescent cell had a cell body bearing a short process orientedsclerally with a bulb-like enlargement (Figure 5A, arrow).Optical sectioning through the cell bodies with confocal mi-croscopy revealed that the fluorescent cells had a large, elon-gated or oval nucleus. The nuclei usually contain 2-3 darkerareas that appear to represent the characteristic dense hetero-chromatin clumps of the cone nuclei in mouse retina [36],which exclude the GFP fluorescence and produce these darkareas (Figure 5B, arrowheads).

© 2003 Molecular VisionMolecular Vision 2003; 9:31-42 <http://www.molvis.org/molvis/v9/a6>

Figure 3. Consistent pattern of GFP expression and the mosaic orga-nization of mouse cones. These are images of fluorescent cones inthe dorsal (A and C) and the ventral (B and D) areas of the retinalwholemounts from one first generation (F1; A and B) and one fourthgeneration (F4; C and D) adult transgenic mouse. They were takenwith an epifluorescence microscope with a 25x lens focusing on thecone cell bodies, showing the consistent dorsal-ventral graded GFPexpression pattern across mouse generations and the appreciablemosaic organization of the mouse cones. Scale bar represents 20 µm.

Figure 4. Fluorescent cells are identifiable on E15. Images werecollected from the dorsal area of a live, unfixed retina of an E15transgenic mouse from line 5933, with the microscope focusing onthe outer surface of the neuroblast layer. A: Pseudocolored (green)epifluorescence microscope image taken with FITC optics, showinga fluorescent cell bearing a process (arrowhead) with bulbous termi-nal enlargement (arrow). B: DIC image, showing the profiles of thesurrounding non-fluorescent retinal cells of the same field. C: Themerged pseudocolor image. Scale bar represents 10 µm.

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To follow the morphological differentiation of the fluo-rescent cells, the developing mouse retinas starting from E17were examined. By E19, the fluorescent cells developed avitreally oriented inner process. The sclerally directed outerprocess became much thicker, it first narrowed down awayfrom the elongated cell body and then expanded into a bul-bous structure, representing a primitive inner segment (Fig-ure 6A). By P2, the inner segments were evident and the axonsbecame thicker (Figure 6B). Apparent outer segment devel-opment appeared consistently in the P4 mouse retina, where aconical structure extended sclerally from the tip of the photo-receptor inner segment (Figure 6C, arrowhead). At this stage,only a few axons had terminal enlargements. The synapticpedicles with apparent fine basal processes were observed insome cones by P7 (Figure 6D, arrowhead). Thereafter, the fluo-rescent cells appeared to be maturing with growing outer andinner segments and enlarging pedicles (Figure 6E,F). An adult-like morphology of the cones basically developed by P10. Thefluorescent cones were fully mature by P20 (Figure 6G), withan overall morphology comparable to that of the adults.

To confirm that the developing fluorescent cells are cones,the cone specific PNA marker [23] was used to stain retinalwholemounts of the early postnatal developing mice. PNAstaining demonstrated that every fluorescent cell in the P1(Figure 7A), P4 (Figure 7B) and P10 (Figure 7C) mouse reti-nas was indeed a cone cell co-labeled by the PNA marker (ar-rows), despite that some cones with PNA staining did not showdetectable GFP (arrowheads). Attempts were made to quan-tify the numbers of PNA positive cones that express GFP dur-ing development. However, accurate counting of the PNApositive cells in the developing retina was problematic, par-ticularly at the early developmental stages, because of the weaksignal, less discrete patchy staining, high background due tobinding of PNA to the interphotoreceptor matrix, inner synap-

tic layer and retinal vasculature during development [23], andthe inability of PNA to reveal the cell morphology. These prob-lems restricted the counting of PNA labeled cones during de-velopment to P10. At this stage, as cones are almost mature,the patchy staining signal of PNA becomes stronger and morediscrete, counting PNA+ cells is less ambiguous. The numberof PNA+ cells expressing GFP is about 91-95% in the dorsaland 32-40% in the ventral of the P10 retina.

© 2003 Molecular VisionMolecular Vision 2003; 9:31-42 <http://www.molvis.org/molvis/v9/a6>

Figure 5. Common morphological features of the E15 fluorescentcells in different mouse lines. These are confocal images of a repre-sentative fluorescent cell in the live E15 retina from another transgenicline 5922. A: 3D image, showing the cell body and a sclerally ori-ented short process (arrow). B: Optical sectioning through the cellbody, revealing that the cell had a typical large, elongated nucleuscontaining 2-3 darker areas (arrowheads) that appear to represent thecharacteristic dense heterochromatin clumps of the mouse cone nu-clei. Scale bar represents 10 µm.

Figure 6. Morphological development of the fluorescent cone cells.These are 3D confocal images of the representative developing fluo-rescent cells taken from retinal wholemounts of the transgenic miceaged at E19, P2, P4, P7, P9, P10 and P20. Apparent outer segmentsbegin to develop on P4 (C, arrowhead). The cone pedicles with finebasal processes appear in some cones by P7 (D, arrowhead). Thecone cells are fully mature by P20 (G). The shadows in these imageswere generated by shadow projections of the corresponding confo-cal Z-stack images for 3D view of the cells. Scale bar represents 10µm.

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Spatial distribution of the fluorescent cones in the mouseretina during development: Figure 8 shows the density pro-files of the fluorescent cones in developing and mature mouseretinas. These density data were from three retinas of 3 miceat each developmental time point. During embryonic stages,the fluorescent cells had a similar ventral and dorsal distribu-tion with peak densities at eccentricity 0.6-1.1 mm. Similardistribution pattern was observed by postnatal day P2. At thisstage, the cone density increased only slightly with peaks ateccentricity 0.9-1.2 mm, although the total number of cones

expressing GFP increased about 1.8 fold due to the expansionof the retinal area. From P2 to P6, the number of fluorescentcones rose steeply in the ventral retina with the highest den-sity occurring at eccentricity 1.2 mm ventral to the optic discand 0.7-1.4 mm dorsal to the optic disc (Figure 8A). A strik-ing feature of the cone density profile in the P6 mouse retinawas the asymmetric distribution of fluorescent cones alongthe ventral-dorsal meridian of the retina, with about 3 timesmore fluorescent cones in the ventral than in the dorsal. FromP6 to P7, the cone density rose only slightly in the ventralretina, but it dramatically increased about 5 fold in the dorsalretina (Figure 8 A). From P7 to P10, the cone density increasedrapidly across the entire retina, but predominantly in the dor-sal retina (about 14 fold in ventral and 23 fold in dorsal; Fig-ure 8A,B). By P10, the density profile of fluorescent coneswas very similar to that of the adult in the ventral retina withpeak densities at eccentricity 1.2-1.4 mm, but reached onlyabout 90% of the adult level in the dorsal retina (Figure 8B).At this stage, the estimated total number of fluorescent conesper retina was about 92% of the adult fluorescent cone num-ber. In the adult mouse, the overall fluorescent cone density inthe dorsal retina is about 3.4 times that in the ventral retina.

© 2003 Molecular VisionMolecular Vision 2003; 9:31-42 <http://www.molvis.org/molvis/v9/a6>

Figure 7. PNA labeling of the fluorescent cells during development.These are confocal images taken from the ventral (A and B) anddorsal (C) areas of PNA stained retinal wholemounts of P1 (A), P4(B) and P10 (C) mice with focusing on the cone inner segments (A)and the outer segments (B and C). Each represents a merged imageof the corresponding GFP (green) and rhodamine (red) images of thesame filed, showing that all fluorescent cells (green) were labeled byPNA (red; arrow) although not every cone expressed the GFP (ar-rowhead). Scale bar represents 10 µm

Figure 8. Fluorescent cone densities in the developing and matureretinas. The average numbers of cones expressing the GFP per mm2

retinal area along the dorsal to ventral meridian through the opticdisc (OD) were counted from montage images of the retinalwholemounts, and plotted against the retinal eccentricity (mm fromthe edge of the OD). Three retinas at each time point were sampledand analyzed.

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The cone densities gradually rose with eccentricity until peakdensities (about 10300 cells/mm2 in the dorsal and 3100 cells/mm2 in the ventral) were reached around 1.2 mm eccentricity,and thereafter the densities fell rapidly toward the far periph-eral retina.

Quantitative analysis of the spatial organization of thecones in adult mouse retina: The mosaic organization of conesvaries widely among vertebrates, even between very closelyrelated species [6,31,38,39]. To quantitatively analyze the spa-tial pattern of cones in the mouse retina, the nearest neighbordistance (NND) analysis was performed. Figure 9 shows theNND distributions of the fluorescent cones in the adult mousecentral (Figure 9A) and far peripheral (Figure 9B) retina. Thedistributions appeared to be symmetric around the mean andfitted well to the normal Gaussian distribution (p>0.10), al-though there was a consistent tail towards larger distances.This indicates a regular distribution of the inter-cone distances,and thus a regular spacing of the cone cells. The average cen-ter to center distance between cone cells was 6.46 ± 1.34 µm(mean and standard deviation) in the central and 8.97 ± 2.53

µm in the peripheral retina. The regularity index (RI), ex-pressed as the ratio of the mean NND to the standard devia-tion, was used to quantify the regularity level of the cone spa-tial patterns; The higher the RI, the more regular the spatialpattern [1,28,29,32]. The regularity index of the cones was4.82 in the central, and 3.55 in the peripheral retina. Theserelatively high RI suggest that the fluorescent cones are orga-nized into a non-random regular pattern, and that the mosaic

© 2003 Molecular VisionMolecular Vision 2003; 9:31-42 <http://www.molvis.org/molvis/v9/a6>

Figure 9. Quantitative analysis of cone mosaic in the adult mouseretina. The histograms represent the distribution of the measuredNNDs of the fluorescent cones in the central (A) and the far periph-eral (B) areas of the dorsal retinas. The lines represent the normalGaussian function, which fitted well to the NND data. “N” is thenumber of fluorescent cones measured. The regularity index (RI) isdetermined by the mean and the standard deviation (s.d.) of the NNDdata, and is not affected by changing the bins of the histograms.

Figure 10. Patterning of the cone mosaic during retinal development.The left panels are representative views of the cone mosaic pattern-ing in the developing mouse retinas. The right panels are the corre-sponding quantitative analysis of the spatial patterns. A-C: Imagesof the fluorescent cones taken from the dorsal retinas of mice aged atE19, P6 and P7, respectively, with an epifluorescence microscope(20x and 10x lens). D,E: Confocal microscope images of the fluo-rescent cones taken from the dorsal retinas of P9 and P10 (E) mice,respectively. The corresponding histograms in the right panels repre-sent the distributions of the measured NNDs of the fluorescent conesin the sampled retinas of the E19, P6, P7, P9 and P10 transgenicmice. The lines represent the normal distribution. The regularity in-dex (RI) did not change significantly from E19 to P6, but increasedfrom P7 to P9 and approached to the adult level at P10. Note thatthere was a consistent tail towards the larger NND in these distribu-tions. Scale bar represents 10 µm.

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array of cones in the central retina is more regular than that inthe far peripheral retina.

Patterning of the cone mosaic during mouse retinal de-velopment: To study the developmental patterning of the

mouse cone mosaic, retinal wholemounts from mice at agesfrom E15 to P10 were examined. Three mice at each age wereexamined and sampled for the NND analysis. From E15 toE18, there were only a few bright fluorescent cells (20-90)per retina, sparsely scattered over both the dorsal and ventralretinal surfaces. On E19, the number of fluorescent cells in-creased significantly. At this stage, these differentiating fluo-rescent cells seemed to be somewhat evenly spaced on theretinal surface (Figure 10A). The distribution of nearest neigh-bor distances of these cells appeared to be symmetric aroundthe mean inter-cell distance (87.86 µm), and closely fitted to abroad Gaussian distribution with a RI of 2.60 (Figure 10, E19).χ2 test of the statistical difference between the normal distri-bution and the actual NND distribution showed no significantdifference (p>0.10). These results indicate that a random dis-tribution is less likely to be the description of the NND data,and that the lower RI might reflect a lower regularity of thepattern. Taken together, these findings suggest that the differ-entiating cones in the mouse retina appeared to be organizedin a non-random array with a lower level of regularity beforebirth. By P6, the spatial arrangement of the fluorescent conesshowed a similar pattern (Figure 10B, P6). On P7, a moreregular mosaic array began to emerge with a higher RI of 3.00and a NND data well fitted to the normal distribution (p>0.10;Figure 10C, P7). On P9, the mosaic pattern seemed to be moreregular with an increased RI of 3.71 (Figure 10D, P9). Anadult-like cone mosaic pattern is formed by P10, where theNND distribution of the fluorescent cones fitted well to thenormal distribution (p>0.10) and the regularity index of 4.56was very close to that of the adult (Figure 10E, P10).

Development and patterning of the cone pedicles duringmosaic formation: To explore whether the formation of thecone mosaic in the mouse retina is associated with cell-cellinteractions via cone synaptic connections, the morphologi-cal development and patterning of the fluorescent cone pedicleswas examined with confocal microscopy. At embryonic stages,the pedicles were not formed. From birth to P6, most of thedeveloping cones had axonal processes with varying sizes ofterminal enlargements. By P7, some cones developed pedicleswith fine basal processes. These pedicles were sparsely dis-tributed in the outer plexiform layer (OPL) of the retina. Conepedicles were observed in more fluorescent cones by P9 (Fig-ure 11A, arrows). At this stage, some cones already had adult-like pedicles with basal processes contacting neighbor cones(Figure 11A, arrowheads), but the patterning of the conepedicles in the OPL was obviously incomplete. By P10, thecone pedicles appeared to be basically mature (Figure 11B,arrows) compared with those of the adults (Figure 11C, ar-rows), and almost every pedicle developed robust basal pro-cesses that contact the neighboring pedicles (Figure 11B, ar-rowheads). The density of the pedicles and the pattern of thepedicle arrays in the OPL were very similar to those of theadults (Figure 11C).

Temporal expression of the mouse cone opsin genes dur-ing development: To test whether the timing of the cone mo-saic development in the mouse retina is temporally correlatedto the appearance of the endogenous mouse cone specific gene

© 2003 Molecular VisionMolecular Vision 2003; 9:31-42 <http://www.molvis.org/molvis/v9/a6>

Figure 11. Developmental patterning of cone pedicles in the outerplexiform layer. These projected confocal images of the representa-tive fluorescent cone pedicles were taken from the dorsal retinas ofthe P9 (A), P10 (B) and adult (C) transgenic mice. The insets wereenlarged images to show the fine processes of the cone pedicles. OnP9, some fluorescent cones exhibited enlarged axonal terminals, whileothers developed pedicles with basal processes (A, arrowhead) thatcontact pedicles (A, arrows) of the neighboring cones. But pattern-ing of the cone pedicles in the OPL was incomplete. The cone pedicleswere well developed by P10, with extensive basal processes (B, ar-rowheads) contacting other cone pedicles (B, arrows). At this stage,the morphology of individual pedicles and the spatial arrays of allpedicles in the OPL were very similar to that in the adult retina (C,arrows: pedicles; arrowheads: basal processes). Scale bar represents10µm.

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expression, the temporal expression of the mouse blue andgreen opsin genes was examined at the transcription level. Inparallel with transgene expression, the strong message of blueopsin gene can be reproducibly detected at E15 (Figure 12),when the first fluorescent cells were identified in the mouseretina. The transcriptional expression of blue opsin gene per-sisted from E15 throughout postnatal stages to adulthood inthe mouse retina. In contrast, the expression of the green op-sin gene was not detectable until postnatal day 7 (Figure 12),when the regular mosaic arrays of cones began to emerge, andthen persisted to adulthood. Younger retinas at earlier devel-opmental stages were also examined. Although a very faintband similar to the blue opsin transcript could be observed atE13-14, unambiguous strong signal that can be reproduciblydetected did not appear until E15. While strong GFP fluores-cence signal was not observed until E15, it is possible that alower level of GFP expression undetectable with theepifluorescence microscope, particularly at the message level,was present in the E13-14 retina.

DISCUSSION The differentiation, morphogenesis and spatial patterning ofmost retinal neurons take place during early developmentalstages. Understanding of these developmental events in conephotoreceptors relies on the development of early appearing,cone specific markers capable of revealing the entire struc-tures of the cone cells. Currently used cone specific antibod-ies, for example, the anti-cone opsin antibodies [24,25,40],have been a valuable tool for identification of the cones. Un-fortunately, these antibodies do not appear to readily stain theearly developing cones in the mouse retina until the cone outersegments begin to develop [26]. Further, these antibodies, likePNA, another commonly used cone marker [23], are unableto label the entire cone cell. While in situ hybridization withcone specific cDNA probes has provided a very useful toolfor studying the photoreceptor patterning in vertebrate retinas

[2,11,12,40], the diffuse hybridization signals often make itdifficult to perform a quantitative analysis on the number andspacing of the cones during mosaic patterning. In addition, insitu hybridization is unable to reveal the morphology of cells.Other markers such as cytochrome oxidase [41] and neuronspecific enolase [26] can also readily label the early postnatalcones. However, these markers are not cone cell specific. Thesetechnical limitations constrain our understanding of the earlyevents of cone development. It is demonstrated in this studythat the transgenic GFP marker can reliably label the develop-ing cones in the mouse retina. In contrast to other cone mark-ers such as PNA, the strong signal of GFP provides a full viewof the entire cone cell morphology in both living and fixedretinas. This allows a reliable measurement of the center tocenter inter-cone distances and unambiguous counting of in-dividual cones for quantitative analysis of the cone mosaic. Inaddition, this transgenic marker also allows the visualizationof the developing mouse cones much earlier than other conemarkers do. These advantages of the GFP marker make it pos-sible for the first time to quantitatively assess the mosaic or-ganization and development of cones in the mouse retina. Sev-eral new observations are made in this study: First, it demon-strates that a subpopulation of cones is organized into a regu-lar mosaic pattern in the mouse retina. Second, it reveals apreviously unidentifiable early phase of mouse cone pattern-ing: a nonrandom array of the cones before birth. Third, itshows that the cone mosaic development appears to follow acharacteristic timetable associated with mouse opsin gene ex-pression and the maturation of cone synaptic pedicles.

One disadvantage in using this transgenic marker, how-ever, is that not every cone in the mouse retina expresses GFP,particularly in the ventral retina. It is obvious that samplingthe GFP labeled cones from the ventral retina would be inad-equate for quantitative analysis of the cone mosaic. To over-come this problem, sampling of the fluorescent cones in thisstudy, was chosen from the central area of the dorsal retina,where the PNA positive cones that express GFP reached 100%in the adult, and about 91-95% in the developing (P10) reti-nas. Thus the quantitative NND analysis of the GFP cones inthis region is a reasonable representation of the native conesin the dorsal retina. Because the cones are fairly uniformlydistributed in both the dorsal and the ventral regions of themouse retina [24,33,40], we speculate that the cone mosaicpattern derived from the dorsal retina also applies to the ven-tral retina. It should be pointed out that those few GFP nega-tive cones, escaped from the NND analysis in the dorsal retina,could increase the NND of a few cones and slightly reducethe regularity of the analyzed patterns by introducing thosetails seen in the NND distributions. Therefore, it is possiblethat the native cone mosaic pattern is slightly underestimatedby analysis of the fluorescent cones in this study. It would beinteresting to simultaneously analyze the NND of PNA posi-tive cells to quantify the mosaic pattern of the native cones inthe mouse retina. Unfortunately, the inability of PNA to readilylabel the cell bodies of mouse cones makes it impossible toaccurately measure the center to center distances of the cellbodies for the quantitative NND analysis.

© 2003 Molecular VisionMolecular Vision 2003; 9:31-42 <http://www.molvis.org/molvis/v9/a6>

Figure 12. Temporal expression of the mouse cone opsin genes. RT-PCR analysis of the developmental expression of the mouse blueand green opsin genes in the mouse retinas. The 504 bp band wasamplified from the mouse green opsin mRNA, and the 351 bp bandwas from the mouse blue opsin mRNA. The first lane is 1 kb DNAmarker.

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Although the transgenic lines had different numbers ofcones expressing GFP, which is presumably due to variationsin transgene copy numbers or position effect variegation, allshared similar dorsal-ventral graded pattern of GFP expres-sion. The parallel temporal expression of the transgene andthe endogenous opsin gene, and the similarity of the spatialdevelopmental pattern of GFP labeling to that of the mousecone opsin staining reported previously [24,40,42,43], indi-cate that the expression of the transgene and the endogenousgenes might be regulated in a similar manner.

Differentiation and morphogenesis of the cones in mouseretina appear to occur in a sequential manner. Most of the earlydifferentiating cones seem to first grow out a sclerally ori-ented process representing the early inner segment and then avitreally descending process before birth. The morphology ofthe fluorescent embryonic cells is consistent with that of theimmature photoreceptors in the embryonic mouse retinas ob-served by Hinds and Hinds [44] with electron microscopy.The location of these fluorescent cells on the outer surface ofthe neuroblast layer and the timing of their appearance duringthe cone genesis, together with the cone specific pattern of thetransgene expression, further suggest that these fluorescentcells are more likely the early differentiating cones. Unfortu-nately, there are hardly any markers available to label the mousecones before birth. Nevertheless, following up the morpho-logical development of the fluorescent cells in early postnatalretinas revealed that these cells had the typical morphology ofmouse cones described in previous studies [23,25,26,42,45-47]. Double labeling with PNA in postnatal mouse retinas start-ing from P1 confirmed that these developing fluorescent cellsare indeed the cones.

The mosaic organization of cones has been documentedin a number of species ranging from fish to primates includ-ing humans [1,2,6,10,12-14]. However, the nature of the conemosaic appears to be species dependent, and varies from highlyregular to irregular [31]. There is a lack of quantitative studyon the spatial organization of cones in the mouse retina. Inthis study, quantitative pattern analysis revealed that the conesin mouse retina are organized in a regular mosaic pattern. Whilethe overall cone mosaic in the mouse retina seems to be lessprecise than that in monkey and cat [1], this less precisioncould be due to the underestimation of the cone mosaic in thisstudy. Nevertheless, the findings in this study suggest that thespatial organization of cones in the mouse retina follows ageneral organization principle shared by cones and other reti-nal neurons in many other vertebrate species, in spite of thefact that mouse is unusual in having only a single type of cone[40] and does not appear to have color vision [21]. In addi-tion, knowledge of this normal pattern of cone organization inthe mouse retina may be useful for examining potential patho-logical alterations in the organization of the cone photorecep-tors in retinal diseases of mutant mice.

Few studies have described the development of retinalmosaic quantitatively. This study provides a quantitative as-sessment of the cone mosaic development in the mouse retina.It appears that the patterning of the cone mosaic in mouseretina is largely late embryonic and early postnatal develop-

mental events with a characteristic timetable. Interestingly, thedistribution of the fluorescent cones appears to exhibit a non-random pattern before the mouse is born, a previously uni-dentifiable early phase of cone patterning. During postnataldevelopment, the peak fluorescent cone densities are clusteredaround the ventral and dorsal centers. There seems to be twodistinct patterns of rapid development of the fluorescent conesaround P6-P7: one occurs in the ventral retina by P6, and theother appears in the dorsal retina starting from P7. In parallelwith the onset of mouse green opsin gene expression and ap-pearance of the immature cone pedicles, the mosaic patternwith higher regularity begins to emerge by P7. Coinciding withthe timing of cone pedicle maturation, the regular pattern ofcone mosaic is basically formed by P10.

It remains unknown in general how a retinal mosaic isdeveloped and what mechanisms control the precision of regu-lar spacing of the mosaic cells. The creation of the mosaicorganization is believed to be a multi-step process of spatiotem-poral neural patterning involving a number of coordinated mo-lecular and cellular events that include cell genesis, cell fatedetermination, activation of the transcriptional network forretinal patterning genes, expression of cell specific markers,cell migration and differentiation [4,5,7,30,48]. The results ofthis study appear to be consistent with the idea of a two phaseprocess of cone mosaic development in the mouse retina. Theearly phase involves the development of a non-random arrayof cones that occurs very early in development. The late phaseis characterized by a gradually increased regularity of the conearrays that reaches the adult pattern when the cone pediclesare maturing. The parallel expression of the transgene and theblue opsin gene at the stage of cone genesis, and the appear-ance of a orderly cone array in the early phase of develop-ment, suggest that the mosaic organization of mouse conesmight be pre-patterned during the embryonic stages. Similarconcept was proposed for mosaic patterning of cones in themonkey retina [49,50]. Since cell migration [30,51], pro-grammed cell death [52] and differential retinal expansion [53]occurring with the developing retina could change the initialpattern of cone arrays, the final mosaic pattern of mouse conesmight require a refining process through certain mechanismsto ensure a regular spacing of cones in the mature mosaic pat-tern. In this sense, the late phase of mouse cone mosaic devel-opment might reflect a refining process of the cone mosaicpattern. Alternatively, the apparent increase in the mosaic regu-larity in this late phase might be due to the increasing num-bers of fluorescent cones at this developmental phase. Never-theless, the remarkable coincidence between the maturationof the mosaic and development of the cone pedicles, suggeststhat synaptic contact mediated cone-cone interactions mightbe involved. Such interactions may feedback to finely tunethe cell positioning through short distance lateral cell move-ment. If it is possible to image the retinas overtime during thelate phase of development, and maintain the integrity of thelive tissue, the GFP reporter in these transgenic mice couldpotentially be valuable in addressing these issues of cone pho-toreceptor development in vivo.

© 2003 Molecular VisionMolecular Vision 2003; 9:31-42 <http://www.molvis.org/molvis/v9/a6>

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ACKNOWLEDGEMENTS This work was supported by NEI grant EY08362 and TheMatilda Zeigler Foundation awarded to Dr. Thomas Hughes.I thank Dr. Hughes for his generous support, encouragementand critical reading of the initial drafts of this manuscript; Drs.Jeremy Nathans and Yanshu Wang for kindly sharing the pR6.5lacZ plasmid; Drs. James Howe and Douglas Gregory for help-ful discussions and reading of the first draft of this manuscript.

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The print version of this article was created on 18 Feb 2003. This reflects all typographical corrections and errata to the article through thatdate. Details of any changes may be found in the online version of the article.