proliferation and expression of progenitor and mature...

Proliferation and Expression of Progenitor and MatureRetinal Phenotypes in the Adult Mammalian CiliaryBody after Retinal Ganglion Cell Injury

Philip E. B. Nickerson,1 Jason G. Emsley,2 Tanya Myers,1 and David B. Clarke1,3

PURPOSE. Despite the identification of a small population ofcells residing in the ciliary body (CB) of the adult mammalianeye that have the capacity to generate retina-like cells in vitro,their activity in vivo remains quiescent. The authors sought toidentify whether the predictable and time-dependent death ofretinal ganglion cells (RGCs) results in activation of progenitor-like cells within the CB.

METHODS. RGC injury was induced by optic nerve axotomy inadult mice. Thymidine-analogue lineage tracing and immuno-cytochemistry were used to identify dividing cells and thephenotype of newly generated progeny.

RESULTS. Two populations of nestin-expressing cells are presentin the CB of the uninjured eye. One population resides inperiendothelial cells of blood vessels, and a second resides inthe ciliary epithelium. Axotomy increases proliferation in theCB, a response that begins before the onset of RGC death andcontinues during a time that corresponds with the peak in RGCdeath. In addition, a subpopulation of nestin-positive cells inthe CB upregulates the homeodomain protein Chx10. Finally,recoverin, the expression of which is normally restricted tophotoreceptors and bipolar cells of the retina, is upregulated inthe CB in a manner that is independent of proliferation.

CONCLUSIONS. Together, these results suggest that progenitor-like cells of the CB respond to cues associated with the loss ofa single retinal cell type and that a subpopulation of those cellsmay differentiate into a cell that bears phenotypic resemblanceto those seen in the retina. (Invest Ophthalmol Vis Sci. 2007;48:5266–5275) DOI:10.1167/iovs.07-0167

The vertebrate retina originates from a population of retinalprogenitor cells (RPCs) in the embryonic primordium of

the diencephalon. During retinogenesis, a temporally choreo-graphed sequence of progenitor proliferation and subsequentinduction of retinal cell phenotypes is initiated. These distinctcellular phenotypes, which constitute all seven neural cell

types of the adult retina, emerge in a well-characterized histo-genic order.1,2 Throughout adulthood, the neurogenic capacityof the vertebrate eye is attenuated; in the mammalian eye, it isvirtually abolished. Continually active postnatal and adult RPCshave been described in the lower vertebrate eye, includingcells located at the border of the retina and adjacent epithe-lium, termed the ciliary margin (CM) of the amphibian eye3 andretinal pigmented epithelium (RPE), rods, and Muller glia infish.4 Cells originating from the CM of the lower vertebrate eyecontinuously generate a population of retinal neurons through-out metamorphosis and adulthood.5 These progenitors thuscontribute to the prodigious capacity for neural regenerationin many lower vertebrates. Some higher nonmammalian verte-brates, such as the postnatal chicken, also retain the capacity torepopulate the adult retina throughout adulthood and underpathologic conditions.6,7

In mammalian systems, the existence of adult retinal neu-rogenic potential was demonstrated using an in vitro colony-forming (neurosphere) assay.8,9 This research identified a smallpopulation of nestin-positive cells derived from the pigmentedciliary epithelium (CE), a bilayer of cells that cover the ciliarybody (CB), a structure involved in mediating lens shape. Thesenestin-expressing cells retain the ability to clonally proliferate,generate sphere colonies, and self-renew for many passages.When exposed to differentiating media conditions, they gen-erate progeny with phenotypes reminiscent of retinal neuronsand glia. These CE cells, termed retinal stem cells (RSCs), havebeen identified in rodent and human eyes.8,9 RSCs differ frommany other neural precursors in that they can proliferate in theabsence of mitogenic factors in vitro and are not abolishedafter the genetic deletion of glial fibrillary acidic protein(GFAP) during development.8,10 In vitro, these cells exhibitmultipotentiality and express transcription factors (Pax6, Six3,Chx10, Rx, Lhx2) and phenotypes associated with other gen-eral precursors in the central nervous system (Nestin,Musashi1, SSEA-1).11 In vivo, mammalian RSCs reside in aquiescent state and show no proliferative activity under con-trol conditions. This quiescence, coupled with the absence ofa putative marker, challenge our ability to characterize anddetermine a role for endogenous RSCs.

In vivo responses of cells in the adult mammalian CB andCM to changes in their intrinsic gene expression have alsobeen investigated. Mutant mice undergoing constitutive activa-tion of the canonical sonic hedgehog signaling pathway displayenriched populations of proliferating cells in the adult CM.12

Cross-breeding of these mutants with a model of retinal degen-eration, a pro23his rhodopsin mutant, generated CM-deriveddivided cells with phenotypes consistent with neurons andphotoreceptors. This evidence suggests that adult CM progen-itors can be stimulated to contribute to the repopulation of theadult mammalian retina.

Responses of adult mammalian CM and CE cells to injuryhave not been fully characterized. Injury to CNS tissue hasbeen shown to generate changes in the local microenviron-ment through the release of diffusible factors and proteinsmediating cell–cell interactions.13–15 Neurogenic regions in

From the Departments of 1Anatomy and Neurobiology and 3Sur-gery (Neurosurgery), Dalhousie University, Halifax, Nova Scotia, Can-ada; and the 2Departments of Neurosurgery and Neurology, MGH-HMSCenter for Nervous System Repair, Harvard Medical School, and Pro-gram in Neuroscience, Harvard Stem Cell Institute, Harvard University,Boston, Massachusetts.

Supported by the Capital Health Research Fund, Halifax, NovaScotia, and the Department of Surgery, Dalhousie University. JGE waspartially supported by fellowships from the Nova Scotia Health Re-search Foundation and the Heart and Stroke Foundation of Canada.

Submitted for publication February 9, 2007; revised April 19 andMay 26, 2007; accepted August 22, 2007.

Disclosure: P.E.B. Nickerson, None; J.G. Emsley, None; T. My-ers, None; D.B. Clarke, None

The publication costs of this article were defrayed in part by pagecharge payment. This article must therefore be marked “advertise-ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: David B. Clarke, Department of Anatomyand Neurobiology, Dalhousie University, 5850 College Street, Halifax,Nova Scotia, Canada, B3H 1X5; [email protected].

Investigative Ophthalmology & Visual Science, November 2007, Vol. 48, No. 115266 Copyright © Association for Research in Vision and Ophthalmology

brain respond aggressively to pathologic conditions broughton by a wide range of damaging stimuli.16–20 In lower verte-brates, a robust neurogenic response in the CM is elicited afterinjury to teleost3 and chicken6 retinas. To a lesser extent, adultmammalian Muller glial cells re-enter the cell cycle and un-dergo reactive gliosis in response to excitotoxic lesions of theretina,21 upregulate appropriate progenitor machinery whenexposed to exogenous growth factors,22 and, in some in-stances, generate neuron-like progeny in vivo23 and in vitro.24

The influence of specific classes of retinal cells on the activityof RPCs during development, including aspects of proliferationand the fate of cells generated by RPCs, has also been de-scribed. Of specific interest is the well-characterized regulationof fate determination during early retinogenesis by accumulat-ing numbers of RGCs.25,26 RGCs are a source of diffusiblefactors that act to regulate progenitor proliferation26 and thefurther production of RGCs.25,26 Recent evidence suggests thatthe proliferative capacity of RPCs is also partially dependent onthe presence of newly generated RGCs.27 The influence ofRGCs on the activity of quiescent neural precursors in the adultmammalian CB, however, has not been described.

The goals of this study were to examine the following in theadult rodent: levels of proliferation within the CB, CM, andadjacent retina; phenotypes of proliferating cells and theirprogeny; and response alterations of these cells after RGCinjury. We describe a novel proliferative and phenotypic re-sponse of cells of the CB to optic nerve (ON) transection, aneffect that is initiated before and increases during the period ofRGC death. Taken together, our results demonstrate that qui-escent progenitors can be activated after selective injury of atleast one class of retinal neuron.

MATERIALS AND METHODS

Balb-c Mice

The use of Balb-c mice (Charles River, St. Constant, Quebec), whichare albino, eliminates pigment autofluorescence and allows for fluo-rescent immunocytochemical phenotyping in the CB. Thirty-day-oldfemale mice were housed two per cage in a colony vivarium main-tained on a 12-hour light/12-hour dark cycle at constant temperature(21°C) and humidity (40%–50%). Food and water were available adlibitum. All animals were cared for by Dalhousie University animal care,following standards described by the Canadian Council for Animal Care

and the ARVO Statement for the Use of Animals in Ophthalmic andVision Research.

ON TransectionThe following methods for ON transection were adapted from thosedescribed in rats.28,29 Briefly, the left eye was transected with mi-croscissors approximately 0.5 mm from the posterior aspect of thesclera. Sham animals underwent the same surgical procedures withouttransection of the ON. Preservation of the blood supply was confirmedby microscopic examination of the retina through the dilated pupil.The right eye of each animal served as an internal, uninjured control.

BrdU Labeling of Proliferating Cells andTheir ProgenyProliferating cells were labeled during the S-phase of mitosis by admin-istration of 5-bromo 2�-deoxy-uridine (BrdU).30 To detect proliferationduring early and later time periods after injury, two labeling paradigmswere used (Fig. 1). In a “pulse” group, three daily intraperitonealinjections of BrdU (0.5 mL, 50 mg/kg, and a dosage shown to consis-tently label proliferating cells in the CNS31,32) were performed for 5days beginning on the day of surgery to detect early postinjury celldivision. A second “chase” group received BrdU in its drinking water(1.5 mg/mL) from the time of injury until killing. Water consumptionwas recorded for individual animals to control for differences in BrdUavailability. Analysis of water consumption showed no significant dif-ferences between individuals and groups (not shown).

Kill and Retinal Tissue ProcessingAnimals were killed 4, 14, and 28 days after surgery (n � 4–5 pergroup). Animals were anesthetized by intraperitoneal injection of alethal dose of sodium pentobarbital (100 mg/kg) and underwent tran-scardial perfusion with chilled solutions of 0.1 M phosphate bufferfollowed by 4% paraformaldehyde in 0.1 M phosphate buffer. Beforethe eyes were removed, sutures were placed in the conjunctiva as areference point for retinal orientation. Eyes and brain were removed,postfixed for 3 hours in 4% paraformaldehyde, and cryoprotected in30% sucrose. Eyes were embedded in gelatin, and the same postfix andcryoprotection procedure was repeated before sectioning (35 �m) ona freezing microtome. Brains, which were used to confirm CNS bio-availability of BrdU, were removed, postfixed in 4% paraformaldehyde,and cryoprotected in 30% sucrose before sectioning at 40 �m. Sectionswere stored in Milonig solution (0.1 M PBS/0.075% sodium azide) untilstaining.

ImmunocytochemistryCellular proliferation and phenotype were determined by fluorescentdouble-and triple-label immunocytochemistry. Tissue was washed in

FIGURE 1. BrdU lineage tracing. Each animal underwent either a pulse or a chase BrdU-labeling regimen to determine the time course ofproliferative responses to RGC axotomy. After transection of the ON, animals were exposed to BrdU (line) and were killed (X) 4, 14, or 28 daysafter injury. Eyes were then examined immunocytochemically to assay the phenotype of dividing cells.

IOVS, November 2007, Vol. 48, No. 11 Retinal Phenotypes after RGC Injury 5267

0.1 M PB for 5 minutes, briefly dipped in ddH2O, and placed in 2 N HClfor 2 hours at room temperature. After washing, sections were placedin blocking solution (8% serum, 0.3% BSA in 0.1 M PBS/3% TritonX-100) for 1 hour at 4°C. Sections were placed in diluent (3% serum in0.1 M PBS/3% Triton X-100) containing primary antibody against BrdUalone, or they were multilabeled using two or more primary antibodies(Table 1). Sections were then rinsed and incubated in either cyanine-conjugated or fluorescent (Alexa; Invitrogen, Carlsbad, CA) second-ary antibodies for 1 hour at room temperature. Sections weremounted onto gelatin-coated glass slides and dried. Fluorescentmounting medium (Vectashield; Vector Laboratories, Burlingame,CA) was used for fluorescent dye–stained (Alexa 488; Invitrogen)sections, and another (Citifluor; Marivac Limited, Halifax, NS, Can-ada) was used for sections stained using Cy2. All antibodies weretested using positive control tissue and primary antibody omission.Nuclei within sections were counterstained (Topro-3 Iodide; Mo-lecular Probes, Eugene, OR).

Confocal Microscopy

Labeled sections were imaged using a confocal microscope (LSM 510or 510 META; Carl Zeiss, Oberkochen, Germany). Objective lenses (1.4

oil/DIC; Plan-Apochromat; Carl Zeiss) ranging from 40� to 100�magnification were used. Pinhole diameters were maintained at 1.0 to2.0 Airy units for all wavelengths when quantifying double- and triple-labeled cells. Laser outputs were set at 5% (488 nm), 80% (543 nm),and 9% (633 nm). Emission filters were 505 to 530 nm (Cy2), 560 to615 nm (Cy3), and more than 650 nm (Cy5). Orthogonal analysis wasused to ensure colocalization of all multilabeled sections.

Cell Counting and Statistics

We used at least four animals per group for our experiments. ForBrdU-labeling experiments, three sagittal sections at the level of theON per animal were visualized on a fluorescence microscope (DM400;Leica, Wetzlar, Germany) equipped with an electronic stepper stage(Ludl, Hawthorne, NY). BrdU-positive nuclei within the CE, CM, andadjacent neural retina were counted and plotted using scientific soft-ware (Neurolucida and Stereo Investigator; MBF Bioscience, Williston,VT). All counting was performed with investigators masked to theexperimental conditions. Unused sections at the level of and adjacentto the ON were used for further phenotypic analysis.

BrdU data are expressed as the mean number of BrdU-positivenuclei per retinal section, at the level of the ON �1.0 SEM. Repeated-

TABLE 1. Antibodies

Antibody Cellular Phenotype Source and Immunizing Information

CRALBP Adult Muller glia 1:1000, Abcam, Cambridge, MACatalog no. ab15051Monoclonal clone B2, IgG2a

GFAP Activated Muller glia and nonretinal 1:100, Novo Castra, Newcastle, UKastrocytes Catalog no. NCL_GFAP_GA5

Clone GA5Species and isotype mouse anti-GFAP IgG1

Glutamine synthetase Adult Muller glia 1:1000, Chemicon, Temecula, CACatalog no. MAB302Species and isotype mouse anti-GS IgG2

Glutamine synthetase whole protein (1–373 bp) used as theimmunizing antigen

Nestin Neuroectodermal, stem/progenitors, RPCs, 1:500, BD PharMingen, San Diego, CAand activated Muller glia Catalog no. 556309

Clone 401Species and isotype mouse anti-nestin IgG1

Musashi-1 Neuroectodermal, stem/progenitors, and 1:1000, ChemiconRPCs Catalog no. AB5977

Species is rabbit anti-musashi-1Pax6 RPCs, amacrine, horizontal, and RGCs 1:500, Covance Research, Berkeley, CA

Catalog no. PRB-278PSpecies is rabbit anti-Pax6

Chx10 RPCs, bipolar, and a small subpopulation 1:500, Chemiconof Muller glia Catalog nos. AB9014 and AB9016

Species sheep anti-recombinant human Chx10Factor 8 Vascular endothelium 1:500, Oncogene Research Products, San

Diego, CACatalog no. PC313Species rabbit anti-factor 8-related antigen/vWF (Ab-1)

DCX Immature, postmitotic neurons 1:500, ChemiconCatalog no. AB5910Species guinea pig anti-DCX

�-III tubulin (Tuj1) Immature and mature neurons 1:1000, ChemiconMouse anti-Tuj1

NeuN Postmitotic CNS neurons 1:1000, ChemiconCatalog no. MAB377Species mouse anti-NeuN

MAP-2 Mature CNS neurons 1:500, ChemiconCatalog no. AB5622Species rabbit anti-MABP-2

Recoverin Photoreceptors and bipolar cells 1:4000, ChemiconCatalog no. AB5585Species rabbit anti-recoverin

BrdU Anti-5-bromo 2�-deoxy-uridine 1:1000, Research Diagnostics Inc., Flanders, NJSheep anti-BrdU

5268 Nickerson et al. IOVS, November 2007, Vol. 48, No. 11

measures analysis of variance was performed, using the independentvariable of surgical group (three levels: transected ON, non-transected ON [right eyes], and sham operated). Repeated-measuresanalysis allowed us to use the right eye as an internal control foreach subject. When faced with a significant main effect, Scheffe andt-test post hoc analyses were performed to test differences amongindividual groups.

To analyze changes in the number of Chx10-positive nuclei in theCE of animals across all groups, we used analysis of covariance, withthe total number of cells in the CE (counts from CE at either end of theretinal section are pooled) of each section as the covariate. Thisanalysis was used to control for differences in the total number ofnuclei within the CE of different sections. Chx10 data are expressed asthe mean number of Chx10-positive cells � SEM, per retinal section.Post hoc t-test comparisons were used to compare differences amonggroups after a significant main effect.

RESULTS

CB and Epithelium Contain Two DistinctPopulations of Nestin-Positive Cells in theUninjured EyeNestin is an intermediate filament protein previously identifiedin adult neural stem/progenitor cells, embryonic neuroectoder-mal stem cells,33 reactive adult astrocytes and radial glia after

injury,34 RSCs and progenitors,35 neurogenic Muller glialcells36 and in floating spheres generated from the CE of adultmice.8,9 Consistent with these in vitro reports, immunocyto-chemical staining of retinas in our in vivo study revealed apopulation of nestin-positive cells in the CB (Figs. 2B–D).Sparsely distributed single nestin-immunoreactive cells werepresent in the CE adjacent to the vitreous (Fig. 2B), consistentwith the nonpigmented layer of the CE. These cells radiate one ortwo thin processes deep to the inner CE, nestin-immunoreac-tive cell population. Nestin-positive cells are also present inepithelium from the peripheral edge of the retina and RPE tothe CB (Fig. 2C). Double-label immunocytochemistry revealedthat nestin-positive cells of the CB do not express GFAP,cellular retinaldehyde-binding protein, or glutamine synthetase(data not shown), lending further evidence in support of pre-vious work reporting that CB stem/progenitor cells do nothave a glial phenotype.10

A previous report indicates that periendothelial cells andpericytes of CNS vasculature express nestin.37 To determinewhether nestin-positive cells in the CB are associated withvasculature, we double-labeled for nestin and endothelial-spe-cific anti–factor 8 (Figs. 2C, 2D). Double labeling with anti–factor 8 revealed two distinct populations of nestin-positivecells. In the deep layers of the CB, nestin colocalized withfactor 8–positive blood vessels (Figs. 2C, 2D), corresponding

FIGURE 2. In uninjured (or control) eyes, two populations of cells of the CB express nestin. (A) Schematic showing the location of the CB. Inset:views represented in micrographs, with the pars plana to the right and the iris to the left. (B) Example of a single nestin-immunoreactive cell (green)found in the superficial epithelial layer of the CB, adjacent to the vitreous. (C) Nestin-expressing cells in the superficial layer of the CB,corresponding to the pigmented CE (arrows and as shown in B), do not stain for anti-factor 8. In contrast, some cells deep to the CE (outlinedin inset and as shown at higher magnification in D) are coimmunoreactive for nestin (green) and endothelial-specific anti-factor 8 (red). Asterisk:blood vessel. (D) Higher magnification of inset in C reveals the presence of nestin on vascular pericytes or periendothelium (arrowheads) and notluminal endothelial cells (arrows). Scale bar: (B, C) 20 �m; (D) 10 �m. Fluorescent dye–stained nuclei are blue in all micrographs.


to pericytes and/or periendothelial cells (high magnification inFig. 2D, arrowhead) and not luminal endothelia (Fig. 2D, ar-rows). Nestin-positive cells in the CE (including individual“displaced” cells and those that form a chain continuous withRPE), however, are distinct from these nestin/anti–factor 8-pos-itive cells of the CB vasculature because of their locationwithin the superficial epithelium of the CB and the absence offactor 8 expression.

Nestin-Positive Cells of the CE Undergo Low-LevelProliferation in Response to RGC Injury

Examination of retinas from sham-operated and right eye, non-injured controls revealed extremely low-level proliferation inthe CB and CM, as seen by BrdU incorporation (typically 1 or2 nuclei per section). Cellular proliferation followed a tempo-ral progression in injured and noninjured retinas. However,proliferation was significantly greater in injured than in unin-jured eyes at 4 and 14 days, when eyes were examined with aBrdU chase paradigm. Differences in proliferation over 14 to28 days were not detectable after a pulse of BrdU, likelybecause of dilution of BrdU by subsequent cell division ordeath. Furthermore, BrdU administration restricted to the firstfew days after axotomy would not be seen because of a delayof several days in CB proliferative response to axotomy.38

Given these observations, data from animals exposed tochronic (chase paradigm; Fig. 1) BrdU exposure are presentedfor the remainder of the study when assessing proliferation.

Interestingly, proliferation increases in the CB of noninjuredeyes over time, suggesting that cells of the CB undergo a

constitutive, slow rate of cell division or that cells recentlygenerated outside the eye migrate to the CB. However, theproliferative response after RGC injury is increased and istemporally restricted: a significant increase in the number ofBrdU-positive nuclei in the CB is seen at 4 days (MEANINJURED

� 1.67, MEANUNINJURED � 0.17; P � 0.003), peaks at 14 days(MEANINJURED � 10.9, MEANUNINJURED � 2.0; P � 0.004), andreturns to levels that are not significantly different from thoseof controls by 28 days (MEANINJURED � 13.7, MEANUNINJURED

� 9.9; P � 0.05) after injury.To determine whether cells of the CB and CE that prolifer-

ate in response to RGC injury express nestin, double-labelimmunocytochemistry was performed. Staining revealed apopulation of recently divided cells in the CE that expressednestin after ON transection (Fig. 3A). Orthogonal confocalanalysis confirmed the presence of BrdU-positive nuclei withinchains of nestin-expressing cells, which themselves extendedto the RPE (Fig. 3B). These dividing cells, however, wererestricted to the pars plicata and did not extend to the parsplana, suggesting that dividing progeny did not migrate to theCM or adjacent neural retina.

Orthogonal analysis was used to quantify the number ofnestin-, BrdU-, and nestin/BrdU-positive cells present in the CEwithin injured eyes (Fig. 3D). Twenty-eight days after injury,when the number of BrdU-labeled nuclei was the highest, 58 �9 cells per section were nestin positive; of those, 27% (16 � 6)were BrdU positive. Conversely, approximately 80% of theBrdU-positive cells in the CB at this time expressed nestin,

FIGURE 3. Nestin-positive cells in the CE proliferate in response to ON transection. (A) Nestin-immunoreactive cells of the CE (green) form achain-like arrangement that is continuous with the RPE (arrows; asterisk delineates the end of the RPE). By 4 days after injury, BrdU-positive nuclei(red) emerge in the CE (arrowheads). Example shown is 28 days after ON transection. Fluorescent dye–positive nuclei are blue. (B) Orthogonalconfocal analysis showing colocalization of BrdU-positive nuclei within a chain of nestin-positive cells. (C) A significant increase in BrdU labeling(mean � SEM BrdU-positive cells per section) is seen at 4 and 14 days after injury compared with controls. By 28 days after injury, BrdU labelingin controls is statistically similar to that in injured eyes (*P � 0.05; significant between injured and uninjured eyes). (D) By 28 days after injury,most (approximately 80%) BrdU-positive cells are nestin positive, indicating that a subpopulation of dividing cells in the CE expresses a progenitorphenotype. Scale bar: (A) 20 �m; (B) 10 �m.


indicating that most recently dividing cells exhibited this pro-genitor-like phenotype.

CE Cells Upregulate Nestin and Chx10 inResponse to Injury

To determine whether progenitor-like cells in the CB respondto retinal injury, we analyzed expression of the transcriptionalregulators musashi-1, Pax6, and Chx10, all of which are phe-notypes expressed in retinal stem cells and RPCs.11 No cells in

the CM or CB expressed musashi-1 or Pax6, with or withoutretinal injury (data not shown). Under control conditions,Chx10 expression was restricted to inner nuclear layer cells ofthe retina (Fig. 4A, arrow); only rarely were Chx10-positivecells found in the CB. After ON transection, however, anincrease in the number of cells expressing Chx10 was evidentin the pars plana and pars plicata of the CB (arrowheads ininjured sections). Analysis of cell counts indicated a significantincrease in the number of Chx10-positive nuclei 4 days (16 �

FIGURE 4. Chx10 is upregulated withinnestin-positive cells in the CE afterinjury. (A) In control and sham-oper-ated retinas, Chx10 (red) immunore-activity is restricted to inner nuclearlayer cells of the retina (arrow forexample; nuclei are blue). ONL,outer nuclear layer; INL, inner nu-clear layer; GCL, ganglion cell layer.Outline of CB in fluorescent dye–stained images. No Chx10 staining isdetected in the CB. At 4, 14, and 28days after injury, Chx10-positive cellscan be seen in the CE (arrowheads).Chx10-positive nuclei appear closeto nestin immunoreactivity (green).(B) Cell counts reveal a significantincrease in the number (mean perretinal section) of Chx10-positive nu-clei in the CE after injury. (C) High-magnification, orthogonal analysisconfirmed colocalization of Chx10-positive nuclei within nestin-positivecells. (D) After injury, the proportionof Chx10-positive cells that also ex-pressed nestin increased relative tocontrol. (B, D) *P � 0.05; significantrelative to control. Scale bar: (A) 50�m; (C) 20 �m.


4; P � 0.02), 14 days (55 � 16; P � 0.02), and 28 days (74 �32; P � 0.05) after injury compared with controls (2.3 � 0.9;Fig. 4B).

Low-magnification observations indicated that Chx10-posi-tive nuclei resided close to nestin-positive cells. Orthogonalconfocal analysis confirmed the coexpression of nestin andChx10 in CE cells (Fig. 4C). Quantification revealed that mostnestin-positive cells expressed Chx10 in their nuclei 4 (85.4%),14 (94.3%), and 28 (90.6%) days after injury (P � 0.01) relativeto controls (16%; Fig. 4D). These data indicate that two previ-ously identified RSC phenotypes (nestin and Chx10) are up-regulated and are expressed by cells in the CE after RGC injury.

Recoverin Is Upregulated in Nondividing Cells ofthe CE after RGC Axotomy

The observation that 20% of BrdU-positive cells in the CB werenot nestin positive (Fig. 3D) prompted us to investigatewhether newly generated, terminally differentiated neuronalphenotypes were present. To examine this possibility, double-label immunocytochemistry for BrdU and either �III-tubulin ordoublecortin was performed. Despite the presence of �III-tubulin–positive cells in the CB (consistent with parasympa-thetic innervation), none of these cells was BrdU positive. Theabsence of doublecortin labeling in all conditions suggestedthat no new neurons were generated. In support of these data,generic postmitotic, mature neuronal phenotypes and RGCs

(MAP-2, NF-200, and NeuN) were also negative (data notshown).

To determine whether BrdU-positive cells generated afterinjury resembled any remaining specific retinal cells, immuno-cytochemistry against a select panel of retina-specific antibod-ies was performed (Pax6 for amacrine cells, PKC� for bipolarcells, GS for Muller glia, calbindin for horizontal cells, andrecoverin for photoreceptors and bipolar cells).39 Under con-trol and uninjured conditions, no cells, including newborn(BrdU-positive) cells, had these phenotypes (data not shown).In contrast, a small population of recoverin-expressing cells(e.g., Fig. 5A, arrow) was seen at 14 and 28 days after injury,suggesting that cells resembling photoreceptors or bipolarcells were present. However, none of these cells was BrdUpositive (Fig. 5), suggesting that nonproliferative cells in the CEupregulate recoverin after injury.

DISCUSSION

Injury-Induced Activation of Progenitor-Like Cellsof the CB

Taken together, our results show that the selective injury of atleast one retinal cell type, the RGC, induces the activation of asmall population of cells in the CB that resemble RPCs. Nestin-expressing cells of the CB proliferate and upregulate Chx10, atranscription factor expressed in active RPCs and RSCs. Finally,

FIGURE 5. Recoverin immunoreactivity is increased in nondividing cells of the CE after retinal injury. (A) Unlike control animals, recoverin-positivecells (green) appear in, and possibly deep to, the bilayered CE as early as 4 days after RGC injury (arrow, for example). Recoverin labeling isintermixed with BrdU-positive nuclei (red; arrowheads, for example). (B) High-magnification, orthogonal analysis confirmed an absence ofcolocalization between recoverin (green) and BrdU (red, arrowhead), indicating that nonproliferative cells upregulate recoverin in response toretinal injury. Scale bar: (A) 20 �m; (B) 10 �m. Fluorescent dye–stained nuclei are blue.


nonproliferative cells present in the CB upregulate recoverin, acalcium sensor protein expressed in photoreceptors and bipo-lar cells, after injury, suggesting an attempt at retinal cellproduction by endogenous precursors.

In this report, we describe a population of cells in the CB ofadult mice that express proteins similar to those seen in retinalstem and progenitor cells previously described by in vitromethods. The influence of the adult retina on the activity ofretinal stem/progenitor cells in the CB has been investigated.Previous reports in lower vertebrates show a salient influenceon adult marginal progenitors located in the peripheral retinaby glucagon-expressing neurons.36 Similarly, the capacity formurine RPCs and Muller glia to enter the cell cycle is influ-enced by retinal tissue.40 These results, supported by thosereported here, clearly show that even into adulthood, neuralprogenitors in the peripheral aspect of the adult eye retain thecapacity to respond to cues provided by the injury or death ofretinal neurons.

Previous studies have identified a number of secreted fac-tors that are released in the retinal microenvironment in re-sponse to RGC injury and death.13 Possible mechanisms ofrelease include recently characterized indirect pathways bywhich Muller glia release a variety of neurotrophic factors inresponse to ON transection.41 In addition, the release of bFGFand glial-derived neurotrophic factor are induced by signalingfrom infiltrating microglia, cells that are present and activeduring RGC death.41,42 The specific mechanism by which CEcells respond to RGC injury is still unclear. Recent evidencesuggests that an increase in proliferation (as measured by Ki67and Cyclin D1 expression) within nestin-positive cells of theCB follows exogenous application of insulin and FGF-2,43 ob-servations that are consistent with those reported in chick44

and that support a role for growth factors in the regulation ofRSC activity.

An unexpected finding evident in our data is the moderateproliferative response detected in retinas 4 days after ONtransection. As discussed, factors released as a function of celldeath can elicit a mitogenic response by CNS precursors. Thisproliferative response 4 days after axotomy, however, pre-cedes the onset of RGC death after axotomy by 1 day,45

indicating that although the proliferative response in the CEmay be augmented by RGC death, it is initiated by injury.

Of particular interest in our study is the activation of tran-scriptional machinery appropriate for progenitor activity afterRGC injury. Chx10 has been shown to play a pivotal role in thedevelopment of the neural retina. Although not necessary forthe genesis of all retinal cell types, mutations in Chx10 result inabnormal eye growth, including microphthalmia, cataracts,and iris malformations.46,47 Furthermore, the absence ofChx10 expression in Chx10 orJ/orJ mice results in small eyesbut a several-fold increase in the number of adult retinal stemcells,8,48 an effect thought to be attributable to the loss innegative regulators elicited by the diminished RPC cell popu-lation. The initial in vitro phenotypic screening during thediscovery of RSCs demonstrated that these cells express nestinand Chx10, two keystone phenotypes of RPCs. Consistent withthese findings, we report in vivo evidence of axotomy-inducedactivation of a population of cells expressing both nestin andChx10 in the same anatomic location. Although this observa-tion is not sufficient to conclude that RSCs are being activated,it is consistent with previous findings.

One well-characterized role of Chx10 is the maintenance ofproliferation within pools of RPCs. Chx10 mutations result inpremature depletion of RPCs and subsequent reduction inretinal volume.46 In our study, we observed a robust increasein Chx10 expression with a relatively modest increase in pro-liferation. Although we would hypothesize that Chx10 upregu-lation should be coincident with proliferation within the CE, it

is possible that its expression is insufficient to elicit a robustresponse in the absence of other mitogenic genes such asPax6, which is not upregulated after RGC injury.

Recoverin Expression in the CB after Injury

We identified a population of cells in the CE that up-regulatethe calcium-binding protein recoverin after retinal injury. Inthe neural retina, recoverin is expressed at relatively earlystages of photoreceptor differentiation49 and in a subpopula-tion of bipolar cells.50 The emergence of recoverin-positivecells in the CE after injury suggests an attempt for the differ-entiation of retinal cell types. CM progenitors in mice can beinduced to generate photoreceptor-like cells in vivo after thecombined overactivation of sonic hedgehog signaling and thepresence of retinal degeneration.12 Given that most recoverin-positive cells in the retina are photoreceptors, one initial spec-ulation might be that an attempt is made toward their genesis.However, previous findings also suggest that cells of the ma-ture bovine CB express components of phototransduction51

and retain the capacity to contract in response to light in theabsence of neural pathways.52,53 In addition, CE and iris tissuescan be induced to generate photoreceptor-like cells in vitro inthe absence of stem cell culture conditions.54 In light of evi-dence supporting a phototransductive ability by CB tissue, itremains possible that RSCs in the ciliary epithelium may rep-resent a residual pool from which young, photoreceptive CEcells may be generated. Specific events leading to the produc-tion of recoverin-positive cells after axotomy appear to involveits upregulation in the absence of proliferation (Figs. 5A, 5B).This observation supports the idea that CE progenitors maythemselves differentiate into a postmitotic cell type indepen-dent of proliferation, an effect reported in subventricular zoneneural precursors after growth factor treatment.55 The at-tempted genesis of an alternative cell type, a bipolar neuron forexample, is also possible because recoverin is present in bipo-lar cells.56

CONCLUSIONS

Our results show that, in vivo, cells of the CB respond toaxotomy with a relatively low level of proliferation that isinitiated before, and increases during, the period of RGC death.In addition, RGC injury increases the number of cells express-ing Chx10 within the CB and the proportion of those Chx10-positive cells that express nestin. Finally, it is evident that cellsof the CE have the capacity to express a phenotypic marker(recoverin) seen in retinal photoreceptors and bipolar neu-rons, a response that is not coincident with cell division. Fromthese data, we conclude that CB cells express phenotypesreminiscent of RPCs and RSCs in response to RGC injury in atime-dependent manner, in accordance with the known tem-poral progression of RGC death. In addition, cells of the CE canbe induced in vivo to express phenotypes normally seen inretinal neurons. Further understanding of the mechanisms un-derlying the activation of RSCs, and their responses to differentpathologic conditions, may provide important insight into thefuture development of cell replacement strategies for treatingvarious retinal abnormalities.

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