Immunohistochemical characterization of the chick marginal ... · Immunohistochemical...

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Braz J Med Biol Res 40(11) 2007

Immunohistochemical characterization of the marginal retina

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Brazilian Journal of Medical and Biological Research (2007) 40: 1455-1464ISSN 0100-879X

Immunohistochemical characterizationof the chick marginal retina

Departamento de Biologia Celular e do Desenvolvimento,Instituto de Ciências Biomédicas, Universidade de São Paulo,São Paulo, SP, Brasil

I.P. Lima*, K. Saito*,D.E. Hamassaki

and C.Y.I. Yan

Abstract

The retina is a highly differentiated tissue with a complex layeredstructure that has been extensively characterized. However, most ofthe previous studies focused on the histology of the central retinawhile little is known about the cellular composition, organization andfunction of the marginal retina. Recent research has identified asubpopulation of multipotential progenitor cells in the marginal re-gions of the retina, closest to the ciliary body (“ciliary marginalzone”). These cells are capable of differentiation in response to anappropriate stimulus. Thus, it is possible that the structure and compo-sition of the marginal retina are distinct from those of the central retinato accommodate the potential addition of newly formed neurons. Tocharacterize the cellular profile of the chick marginal retina, welabeled it immunohistochemically for markers whose staining patternis well established in the central retina: calbindin, calretinin, proteinkinase C, and choline acetyltransferase. Calbindin was present at verylow levels in the marginal retina putative photoreceptor layer. Calret-inin-positive horizontal cells were also sparse close to the ciliarymarginal zone. The bipolar cells in the marginal outer plexiform layerwere positive for anti-protein kinase C antibodies, but the density oflabeling was also decreased in relation to the central retina. In contrast,the marginal starburst cholinergic amacrine cell pattern was verysimilar to the central retina. From these data we conclude that thestructure of the marginal retina is significantly different from that ofthe central retina. In particular, the expression of late retina markers inthe marginal retina decreased in comparison to the central retina.

CorrespondenceC.Y.I. Yan

Departamento de Biologia Celular

e do Desenvolvimento

ICB, USP

Av. Prof. Lineu Prestes, 1524

Sala 407

05508-900 São Paulo, SP

Brasil

Fax: +55-11-3091-7402

E-mail: [email protected]

*These authors contributed

equally to this study.

Research supported by FAPESP

(No. 01/09047-2). I.P. Lima was

the recipient of a CAPES graduate

research fellowship.

Received November 28, 2006

Accepted August 2, 2007

Key words• Marginal retina• Ciliary marginal zone• Calbindin• Calretinin

Introduction

The vertebrate retina plays an importantsensory role, conveying visual input to higherlevels of the nervous system. Its highly dif-ferentiated multilayered structure reflects thephysiological complexity required for such arole. The fulfillment of this sensory function

takes place mainly in the central region.Accordingly, this region of the retina hasbeen extensively characterized, both in termsof cellular distribution and of the expressionof critical markers. In contrast, the cellulardistribution and laminar organization of themarginal retina are still poorly understood.The marginal retina is situated next to the

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lens and ciliary body and it differs from thecentral retina in various functional and mor-phological aspects, such as visual field, cel-lular subtype distribution and density (1-3).Finally, the differentiation of the retina dur-ing oculogenesis occurs in a central-periph-eral wave, so that the marginal retina ma-tures later during development (4,5). Whilein the central retina cells undergo mitoticarrest and differentiation early during devel-opment (E5 in chicks) (6), the marginal retina,which is located 100 µm from the edge of theretina, contains proliferating cells even inthe adult animal. The mitotic activity ofthese cells was indirectly identified by theirability to incorporate the thymidine analogbromodeoxyuridine (BrdU) during DNA rep-lication. This proliferating region has beennamed ciliary marginal zone (CMZ) (7,8).

In chicks, specifically, it has been shownthat the proliferating cells of the CMZ cangenerate amacrine and ganglion cells in theabsence of exogenous stimulation (7). Fur-thermore, upon exogenous application ofinsulin and FGF2, they can also expressmarkers for amacrine, horizontal, photore-ceptor, and ganglion cells (8,9). Lastly, theseprogenitor cells increase their proliferationupon acute retinal damage (9). Taken to-gether, these data suggest that, similar tolower vertebrates, the progenitor cells of thechicken CMZ are multipotent under adequateconditions and potentially participate in reti-nal regeneration.

In light of this possibility, further identi-fication of the cellular components of themarginal region and their stratification wouldaid our understanding of the mechanismsinvolved in the response of the marginalretina to retinal damage. The accommoda-tion of newly differentiated CMZ cells intothe existing marginal network suggests amore plastic histological organization of thisregion, possibly distinct from the centralretina and which might illustrate the se-quence of events necessary for retinal regen-eration. Moreover, knowledge of the histo-

logical organization of the marginal retinacould facilitate the identification of the newlydifferentiated cellular subtypes through theirlaminar localization, as is done in the centralretina.

In the present study, we histologicallycharacterized the marginal retina, includingthe CMZ, using key markers for central retinacell types (photoreceptors and horizontal,bipolar, amacrine, and ganglion cells): cal-bindin, calretinin, protein kinase C (PKC),and choline acetyltransferase enzyme (ChAT).The patterns for these markers were com-pared with previously characterized centralpatterns to establish, through their expres-sion, the equivalent marginal cell layers.

Material and Methods

Animals

One-day-old (P1) Leghorn chicks (Gallusgallus, obtained from Kunitomo Chicken Farm,Mogi das Cruzes, SP, Brazil) were maintainedon a 12-h light/dark cycle until P5, when theywere sacrificed around 10:00 am with an over-dose of ketamine (30 mg/100 g body weight)and xylazine (6 mg/100 g body weight), andtheir eyes were rapidly removed. After cuttingaway the anterior pole or the posterior polesfor central or marginal retina experiments, thevitreous humor was removed and the eyeswere immersed in ice-cold 2-4% paraformal-dehyde in 0.1 M phosphate buffer (PB) at pH7.4 for 3 h. The tissue was then cryoprotectedin 30% (w/v) sucrose for at least 48 h, frozen inembedding medium (O.C.T., Sakura Finetek,Torrance, CA, USA), cut perpendicularly tothe vitreal on a cryostat (12-µm transversesections), and collected on gelatin-coated glassslides.

These experiments were approved by theEthics Committee for Animal Research ofthe Biomedical Sciences Institute, Univer-sity of São Paulo (protocol number 052),and were conducted in accordance with NIHguidelines.

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Bromodeoxyuridine detection

Chicks were anesthetized with ketamine(5 mg/100 g body weight) and xylazine (1 mg/100 g body weight) and 20 µL BrdU (10 µg; 5-bromo-2-deoxyuridine; Sigma, St. Louis, MO,USA) was injected in the right eye at P1, P2,and P4. The contralateral control eye (left)was injected with 20 µL saline. These chickswere sacrificed at P5 and processed as men-tioned above. Retinal sections were incubatedin 2 M HCl for 60 min at 37ºC for DNAdenaturation, washed three times in PB, andblocked with 3% normal goat serum, 1% bo-vine serum albumin and 0.5% Triton X-100 inPB for 30 min. They were then incubatedovernight with rat anti-BrdU primary anti-body (Accurate Chemicals, 1:200, Westbury,NY, USA) in 0.5% Triton X-100 and 3%normal goat serum in 0.1 M PB. After exten-sive washes in 0.1 M PB, sections were incu-bated with anti-rat Alexa-488 secondary anti-body (1:1000, Molecular Probes, Eugene, OR,USA). After several washes in PB, slides werecoverslipped using Vecta Shield (Vector Labo-ratories Inc., Burlingame, CA, USA). Theresults were analyzed with a Nikon PCM2000(Melville, NY, USA) confocal microscope.The control sections without the primary anti-body were not labeled (data not shown).

Immunohistochemistry

The following primary antibodies wereused: anti-calretinin (1:500, Chemicon AB1550, Temecula, CA, USA) (10,11), anti-ChAT (1:100, Chemicon AB144P) (11), anti-calbindin (1:200, Sigma C8666) (1) and anti-PKC-α (1:1000, Sigma P4334) (12,13). Reti-nal sections were incubated with the primaryantibodies overnight and with Alexa-488 orTRITC-conjugated secondary antibodies(1:1000 or 1:200, respectively; MolecularProbes) for 2 h. Propidium iodide labelingwas performed after the secondary antibodywashes. The slides were treated with RNAseA (10 mg/mL; Sigma) for 30 min and incu-

bated with 20 mg/mL propidium iodide (Sig-ma) for 30 s. The slides were then washedand coverslipped with Vectashield and ana-lyzed with a Nikon PCM2000 confocal mi-croscope.

In addition to anti-calbindin, fluorescentlylabeled peanut agglutinin (PNA; Vector Lab-oratories, FL-1071 at 1:50 dilution) was alsoused as a cone-specific marker (14,15).

Results

The marginal retina encompasses theCMZ, which contains a population of prolif-erating progenitor cells. To identify theseproliferating cells in the marginal retina andto determine the size of the CMZ, we de-tected the incorporation of the thymidineanalog BrdU. BrdU was injected intraocu-larly and its presence detected by immuno-fluorescence (Figure 1D,E). Confirming pre-vious reports, BrdU-positive cells were con-centrated in a region 100 µm from the edgeof the retina where it meets with the retinalpigmented epithelium (7). There were noBrdU-positive cells in the central retina (datanot shown). Thus, for our analysis, we de-fined the CMZ as the peripheral-most 100-µm stretch inside the retina that clearly con-tains proliferating cells. To compare betweenthe CMZ and its surrounding non-proliferat-ing region, we also analyzed a region ex-tending 250 µm into the retina from theinterface of the retina and the ora serrata. Werefer to this region as the marginal retina,which includes the CMZ. Finally, the centralretina was defined as the dorsotemporal areaabove the pecten.

For an initial comparison of the layerspresent in central and marginal retinas, weused propidium iodide, a nuclear marker(Figure 1A-C). As expected, in the centralretina the nuclear layers alternated with plexi-form layers (Figure 1A). In the marginalregions, the ganglion cell, inner nuclear andplexiform layers continued from their re-spective layers observed in the central retina

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Figure 2. Calbindin-positive cellsin central and marginal retina. A,Central retina; B, temporal mar-ginal retina; C, nasal marginalretina. ONL = outer nuclearlayer; INL = inner nuclear layer;GCL = ganglion cell layer. Thevitreous surface faces the bot-tom of the figure. Arrowheads in-dicate beginning and end of theciliary marginal zone. Scale barsin A = 40 µm and in B and C = 50µm.

but, as soon as they approached the CMZ,these layers narrowed (Figure 1B,C). Theouter nuclear layer was interrupted and re-duced, and the outer plexiform layer practi-cally disappeared at the ciliary-most mar-ginal region. In addition, cells in the outer-most layers presented a fusiform shape ratherthan the more spherical shape observed inthe central retina. This initial characteriza-tion indicated that the marginal retina ishistologically distinct from the central retina,with a decrease in fibers and modificationsin cell shape.

To determine whether the relative contri-bution and distribution of the different celltypes in each nuclear layer also change, weanalyzed in the marginal retina the expres-sion pattern of different central retinal cellmarkers that characterize subgroups of reti-nal cell types.

Calbindin

Confirming previous studies (10,16),calbindin in the central retina was predomi-nantly found in photoreceptors, and in theinner nuclear (bipolar and amacrine cells)and ganglion cell layers (Figure 2A). In themarginal retina furthest from the CMZ, thedistribution of calbindin-positive cells in theputative marginal inner nuclear layer wassimilar to that of the central retina (Figure2A-C). However, labeling at the ganglion

Figure 1. Central and marginal retinal histology. A-C,Propidium iodide nuclear staining. A, Central retina; B,temporal marginal retina; C, nasal marginal retina. ONL= outer nuclear layer; INL = inner nuclear layer; IPL =inner plexiform layer; GCL = ganglion cell layer. D andE, Proliferating cells in the retinal ciliary marginal zonelabeled with bromodeoxyuridine. D, Nasal marginalretina; E, temporal marginal retina. The vitreous sur-face faces the bottom of the figure. Arrowheads indi-cate beginning and end of the ciliary marginal zone.Scale bar = 20 µm in all panels.

A B

C

D E

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cell layer of the CMZ was increased in inten-sity (Figure 2C). In contrast, when approach-ing the CMZ, the intensity of calbindin ex-pression in the outer nuclear layer decreasedsignificantly (Figure 2B and C). The nasaland temporal retinas differed in the extent ofdecrease. Specifically, calbindin immunore-activity extended further into the ciliary bor-der in the nasal retina than in the temporalretina: while in the nasal retina the absenceof calbindin-positive cells occurred near theCMZ, in the temporal marginal retina, thelabeling for photoreceptors decreased muchearlier (compare Figure 2B and C).

To further analyze the differences be-tween central and marginal photoreceptors,we also used the lectin PNA, which labelscone-associated matrix, primarily the outerand inner segments of the cone (17). Similarto the calbindin-labeling pattern in the mar-ginal retina, the binding of PNA in this re-gion was very weak (data not shown).

Protein kinase C-ααααα

In the central retina, PKC has been lo-cated in bipolar cell bodies and their den-drites and axons going to the outer and innerplexiform layers (IPL), respectively (Figure3A). In the IPL, the immunolabeling ap-peared in a punctiform pattern (Figure 3B,C).

The labeling of the inner nuclear layerextended until the CMZ both in the temporaland nasal marginal retina (Figure 3B,C).The main differences in PKC immunoreac-tivity were found in the outer plexiformlayer, which became faint and discontinuousin both the nasal and temporal CMZ. Inaddition, in the inner nuclear layer of themarginal regions furthest from the CMZ,PKC was detected both in cell bodies andbipolar projections. The PKC-containing cellbodies were mostly concentrated in the in-nermost portion of the inner nuclear layer,whereas in the central retina, they werelocated preferentially in the external por-tions.

A

B

C

Figure 3. Identification of proteinkinase C-α-positive bipolarcells. A, Central retina. The ar-row points to a labeled centralbipolar cell; B, temporal mar-ginal retina. The arrow points toa labeled marginal cell that emitsprocesses towards the plexiformlayer; C, nasal marginal retina.OPL = outer plexiform layer; INL= inner nuclear layer; IPL = in-ner plexiform layer; GCL = gan-glion cell layer. The vitreous sur-face faces the bottom of the fig-ure. Arrowheads indicate begin-ning and end of the ciliary mar-ginal zone. Scale bars in A = 20µm and in B and C = 50 µm.

Calretinin

Besides labeling in horizontal cells, cal-retinin labeling was observed in amacrineand ganglion cells of the central retina, aspreviously described (Figure 4) (7,10). Al-though we detected calretinin immunoreac-tivity in the putative marginal amacrine cells,labeling of horizontal cells decreased sig-nificantly when approaching the CMZ. Adecrease in labeling was also seen in theirprocesses in the outer plexiform layer.

An additional difference between centraland marginal retinas was a decrease in label-ing in the IPL. Whereas the central IPL wasstrongly labeled, labeling in the marginalretina IPL was weak and punctate (compareFigure 4A, B and C). In the CMZ even thissparse labeling in the IPL disappeared. In the

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ganglion cell layer a similar but less pro-nounced decrease in calretinin staining wasobserved towards the CMZ (Figure 4B,C).

Choline acetyltransferase

Confirming the results of Spira and col-laborators (18), ChAT was detected in ama-crine cells located in the inner nuclear layerand in the ganglion cell layer of the centralretina, branching into two bands of the IPL(Figure 5A).

In the marginal retina closest to the cen-tral region, ChAT-immunolabeling wasfound in cells with morphology similar tothat of the central amacrine cells, and inprocesses in the IPL (Figure 5A-C). In theCMZ, however, the two ChAT-positive bandswere no longer parallel but converged, andamacrine cells of the inner nuclear layer andganglion cell layer in the CMZ approachedeach other as well (Figure 5B,C). This con-vergence most likely is due to a gradual reduc-tion of the layers that results in the thinning ofthe retina as it nears the ora serrata.

Discussion

Previous studies have extensively ana-lyzed the histology of the central retina,demonstrating that this structure is dividedinto distinct layers composed of cell bodiesand processes. Similarly, the marginal retinaalso displays distinct layers, each with acharacteristic cellular distribution. However,our data show that in the marginal retinasome layers are modified relative to the cen-tral retina. The differences between the cen-tral and marginal retina are summarized inTable 1 and were mainly found in the outer-most layers. For example, in the marginalretina there was a reduction in the labelingfor calbindin and PNA in photoreceptor lay-ers. Moreover, the PKC immunoreactivityof bipolar cell dendrites in the outer plexi-form layer and calretinin-positive horizontalcells disappears in the marginal zone.

Figure 4. Comparison of cal-retinin-positive cells in centraland marginal retina. A, Centralretina. The arrow points to a la-beled central horizontal cell; B,temporal marginal retina; C, na-sal marginal retina. OPL = outerplexiform layer; INL = inner nu-clear layer; GCL = ganglion celllayer. The vitreous surface facesthe bottom of the figure. Arrow-heads indicate beginning andend of the ciliary marginal zone.Scale bar = 20 µm in all panels.

Figure 5. Choline acetyltrans-ferase-immunopositive cells. A,Central retina; B, temporal mar-ginal retina; C, nasal marginalretina. INL = inner nuclear layer;GCL = ganglion cell layer. Thevitreous surface faces the bot-tom of the figure. Arrowheads in-dicate beginning and end of theciliary marginal zone. Scale barsin A = 10 µm and in B and C = 15µm.

A

B

C

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Calbindin is mainly associated with cen-tral cone photoreceptors and amacrine cellsin diurnal animals (19-21). Here, we showthat calbindin-labeling was absent in theputative photoreceptor layer of the chickmarginal retina. An explanation would bethat the cones in the marginal zone might notbe sufficiently differentiated to express cal-bindin. Calbindin is first seen in photorecep-tors of the central retina in E16 chick em-bryos (16). Moreover, previous studies haveshown that the temporal CMZ contains moreproliferating progenitor cells than the nasalCMZ (7), which means that the temporalmarginal zone potentially contains more un-differentiated cells. Thus, if calbindin is amarker of the final stages of photoreceptordifferentiation, the calbindin-negative mar-ginal photoreceptor layer should be moreextensive in the temporal retina than in thenasal retina. Accordingly, we observed thatthe temporal marginal photoreceptor layerlost calbindin-labeling much farther fromthe CMZ than the nasal marginal retina.Therefore, one explanation for the calbindin-negative marginal photoreceptor layer couldbe that this region is populated with photore-ceptors that are not yet terminally differenti-ated. Retardation in marginal photoreceptordifferentiation could also explain the de-crease in PNA binding in the putative mar-ginal photoreceptor layer. Other lines of ev-idence support this possibility. For instance,photoreceptor oil droplets appear first in thecenter and later in the periphery (22), and thedevelopmental timing and distribution ofwavelength-specific cone opsin expressionalso differs from center to periphery (1).

An alternative explanation is that themarginal retina - especially closer to theCMZ - is maintained in an undifferentiatedstate. This possibility is supported by a de-tailed analysis of the CMZ of older post-natal chicks (7). Neither calbindin nor visininwas detected in the outer nuclear layer ofthis region even in P14 animals. In contrast,cell proliferation in the CMZ is maintained

at least up to P19, suggesting that the adultCMZ is optimized for the maintenance ofundifferentiated proliferating cells and there-fore does not support differentiation. In thisscenario, daughter cells of the CMZ pro-genitor pool would have to leave the CMZenvironment to express markers of a moredifferentiated state. This hypothesis is sup-ported by cell fate experiments. Proliferat-ing cells from the CMZ migrate away fromthe margin and are sometimes found in thegranular cell layer and outer nuclear layer ofthe non-proliferating marginal retina (8,23).Cell-type specific markers begin to appearwhen the cells are farther from the CMZ(7,24).

Finally, another explanation for the lackof calcium-binding proteins at the peripherywould be that marginal cones have lowerintracellular calcium levels. The immunore-activity of calbindin, calretinin and parval-bumin depends on the occupancy of theircalcium-binding sites. These proteins arebetter recognized by their respective anti-bodies in the presence of calcium (25). Thus,

Table 1. Intensity of immunolabeling in central and marginal retinas.

Calbindin Calretinin ChAT PKC

ONLCentral + - - -Marginal - - - -

OPLCentral - - - ++Marginal - - - +

INLCentral +++ +++ ++ +++Marginal +++ ++ ++ +++

IPLCentral ++ ++ ++ +++Marginal ++ ++ ++ +++

GCLCentral ++ ++ ++ +Marginal ++ ++ ++ +

The intensity of calbindin, calretinin, choline acetyl transferase (ChAT), and proteinkinase C (PKC) labeling was assessed qualitatively for the outer nuclear layer (ONL),outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), andganglion cell layer (GCL). + indicates intensity of labeling; - indicates absence oflabeling.

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the decrease in marginal labeling for calbin-din could be caused by diminished intracel-lular calcium levels and not by a decrease incalbindin itself.

Differences in the histology and immu-nolabeling pattern of the marginal photore-ceptor layer, irrespective of the cause, sug-gest that additional marginal retinal compo-nents could also be distinct from those in thecentral retina. This difference should be re-flected in the pattern of immunolabeling forother retina cell type markers associated withthe inner layers. To assess this point, weemployed antibodies against PKC-α, cal-retinin, and ChAT, which are respectivelymarkers for bipolar, horizontal, and ama-crine cells.

Particularly striking was the disappear-ance of PKC and calretinin immunoreactiv-ity in the outer plexiform layer. Our datashowed that the PKC-positive cells near theCMZ extended neurites towards the IPL butnot towards the outer plexiform layer. Incontrast, still in the marginal retina, but fur-ther from the CMZ, the PKC-positive cellscould be seen extending their processes bidi-rectionally. This pattern is similar to thatseen for early bipolar cell development inchick and rat retina: PKC-positive bipolarcells extend neurites first towards the IPLand then establish connections in the outerplexiform layer (26,27). Thus, if we con-sider the dynamics of PKC immunoreactiv-ity in bipolar cell neurites as an indicator ofthe state of differentiation of the bipolar cell,the data presented here suggest that they notonly are reduced in number in the marginalregion, but are also relatively undifferenti-ated.

Our results with labeling for calretininalso suggest that marginal horizontal cellsdiffer from central ones. Calretinin expres-sion in chick central horizontal cells is onlydetected from embryonic stage 18 (E18)onwards (16). In the marginal region, calret-inin-positive cells in the outermost region ofthe inner nuclear layer were absent in the

neighborhood of the CMZ, suggesting thatputative horizontal cells of this region arenot sufficiently differentiated to express cal-retinin. Conversely, calbindin expression inchick central amacrine cells is present byE10 in the innermost part of the inner nu-clear layer (16). In our experiments calbin-din labeling was detected throughout thecorresponding region in the marginal retina.Taken together, these data suggest that theputative inner nuclear layer of the CMZexpresses markers that are present early inthe central retina such as calbindin, but notlate markers such as calretinin.

The hypothesis that the marginal zonecontains a histologically distinct subpopula-tion of cells that are still undergoing differ-entiation is supported by observations in fishand frog retina. Both are capable of retinalneurogenesis in the marginal zone through-out their lives, and their marginal cells dis-play a periphery-central-directed gradient ofgene expression that correlates with the dif-ferentiation status, with more marginal cellsexpressing markers of earlier states of dif-ferentiation (23,28,29). An equivalent pro-liferative zone was not found in mammalianCMZ (30). This absence could be due to anevolutionary decrease of the Shh pathwayactivity in the retina (31). Instead, a prolif-erative population was found in the mamma-lian pigmented ciliary margin (32,33). Simi-lar to amphibian and avian CMZ cells, thesemammalian adult retinal progenitor cells canalso express different retinal markers afterarresting from the cell cycle, rendering thema highly promising tool for retinal regenera-tive therapies.

For targeted regenerations, it would beconvenient to control the rate of retinal pro-genitor proliferation and to direct their dif-ferentiation pathway. In chicks, intraocularinjections of insulin and fibroblast growthfactor stimulate proliferation at the CMZ(7). This same treatment induces differentia-tion of progenitors but mostly into amacrineand ganglion cells (9). Conversely, neural

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progenitor cultures derived from mouse cili-ary body can be induced in vitro to express alarger variety of cell markers, including pho-toreceptors and bipolar and Müller glia mark-ers (32,33). This difference in potentialitybetween in vivo and in vitro settings suggestthat the retinal marginal microenvironmentmight contain elements that restrict the mul-tipotentiality of the adult progenitor cells.Thus, although significant progress has beenmade in our understanding of vertebrate adultretina progenitors, we believe that furtherinsight into the composition of this regionwill contribute to our understanding of the

fate of the multipotential progenitor cells, aswell as the connective plasticity required forincorporating them into the existing neuralnetwork.

Acknowledgments

The authors would like to thank AndréaS. Santos Bredariol for excellent technicaladvice, and Dora Ventura and Daniela Bonci(Department of Experimental Psychology ofthe Universidade de São Paulo, SP, Brazil)for sharing the PKC antibody.

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