michael a. dyer and constance l. cepko- control of müller glial cell proliferation and activation...

8/3/2019 Michael A. Dyer and Constance L. Cepko- Control of Müller glial cell proliferation and activation following retinal inj…

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Glial cells are found ubiquitously throughout the central nervoussystem (CNS), where their pr imary role is to maintain neuronal

health. When CNS homeostasis is perturbed as a result of trauma,neurodegeneration, inflammatory disease or excitotoxicity, glialcells become activated and un dergo reactive gliosis1–6. This

process is characterized by proliferation and changes in geneexpression that are believed to be important for the protection

or repair of neurons1–5

. In addition to the benefits ascribed toglial–neuronal interactions, glial cell activation is proposed to beharmful under certain circumstances, and to contribute to dis-

ease progression1,4.The vertebrate retina contains a specialized type of glia, the

Müller glia, not found elsewhere in the CNS. Like other glial cellsof the CNS, Müller cells undergo reactive gliosis following acuteretinal injury or chronic neuronal stress7–9. Müller cell gliosis is

characterized by proliferation8, changes in cell shape due to alter-ations in intermediate filament production10, changes in iontransport properties11, and secretion of signaling molecules such

as vascular endothelial growth factor12,13. Ostensibly, gliosis isimportant for the protection and repair of retinal neurons, yet

some pathologies such as diabetic retinopathy may be exacer-bated by reactive gliosis11–13. Although a great deal is known about

the environmental factors that can induce glial cell activation andthe changes in these cells during gliosis, very little is known abouthow the process is regulated. We were particularly interested inthe regulation of Müller glial cell proliferation and the changes

in cell shape that result from the rapid upregulation of glial fib-rillary acidic protein ( GFAP) following acute retinal injury

because these are the earliest hallmarks of reactive gliosis8,14, andthey have been documented in a variety of neuropathologicalconditions of the CNS15–19.

The molecular events that lead to cell cycle re-entry from aquiescent G0 state are extensively characterized in tumor cells andfibroblasts20–22. Phosphorylation of the retinoblastoma protein

(Rb) or related family members by a cyclin/cyclin-dependent-kinase (CDK) complex leads to de-repression of transcription of

E2F-regulated genes and entry into S-phase21–23. Cyclin kinaseinhibitors (CKIs) of the Cip/Kip family regulate cell-cycle pro-

gression by forming a ternary complex with the cyclin/CDK com-plex, thereby blocking phosphorylation of Rb24.

We have found that the p27Kip1 cyclin kinase inhibitor interacts

with cyclin D3 in Müller glial cells of the adult mouse retina. Fol-lowing retinal injury, p27Kip1 protein levels decrease, and Müller

glial cells re-enter the cell cycle and upregulate GFAP. Shortlythereafter, cyclin D3 is downregulated. This latter event m ostlikely is what prevents deregulated glial cell proliferation follow-

ing acute or chronic retinal injury. The changes in p27Kip1 expres-sion are not simply correlated with reactive gliosis. Examination

of a p27Kip1 knockout mouse revealed gratuitous Müller glial cellactivation and GFAP upregulation shortly after these cells wereformed during retinal development. The p27Kip1-deficient mice

also exhibited retinal dysplasia, which is suggested25 to be due toouter limiting membrane disruptions that most likely result fromwidespread glial cell activation during development, rather than

from photoreceptor proliferation. In addition, dramatic alter-ations in the retinal vasculature were observed in retinae con-

taining Müller glial cells undergoing reactive gliosis. This findingsupports the hypothesis that Müller glial cell activation is impor-

tant in the progression of diabetic retinopathy.

RESULTS

Cyclin D3 and p27Kip1 are expressed in M üller gliaMüller glial cell bodies lie in a narrow band in the middle of theinner nuclear layer of the adult retina (Fig. 1a). Their processes

span all cellular and plexiform layers of the retin a, formingmicrovilli at the apical surface26. We found that the p27Kip1 cyclinkinase inhibitor was expressed in a restricted row of nuclei in the

middle of the inner nuclear layer, consistent with Müller glial celllocalization (Fig. 1b). To directly demonstrate that Müller gliaexpressed p27Kip1, we did co-immunolocalization using an anti-

body directed against cellular retinaldehyde-binding protein(CRALBP; Fig. 1c). Nuclei that were immunoreactive for p27Kip1

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Control of M üller glial cellproliferat ion and act ivat ion

following ret inal injury

Michael A. Dyer and Constance L. Cepko

Department of Genetics and Howard Hughes Medical Institute, Harvard Medical School, 200 Longwood Avenue, Boston, Massachusetts 02115, USA

Correspondence should be addressed to C.L.C. ([email protected])

Müller glial cells are the major support cell for neurons in the vertebrate retina. Following neuronaldamage, Müller cells undergo reactive gliosis, which is characterized by proliferation and changes ingene expression. We have found that downregulation of the tumor supressor protein p27Kip1 and re-entry into the cell cycle occurs within the first 24 hours after retinal injury. Shortly thereafter, M üllerglial cells upregulate genes typical of gliosis and then downregulate cyclin D3, in concert with anexit from mitosis. M ice lacking p27Kip1 showed a constitutive form of reactive gliosis, which leads toretinal dysplasia and vascular abnormalities reminiscent of diabetic retinopathy. We conclude thatp27Kip1 regulates Müller glial cell proliferation during reactive gliosis.

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precisely colocalized within the CRALBP-immunoreactive Müllerglial cell bodies (Fig. 1d and e). Cyclin D3 is expressed in thenuclei of Müller glial cells (C. Ma and C.L.C., unpublished data).

Co-immunolocalization using antibodies directed against cyclinD3 and p27Kip1 revealed that Müller glial cell nuclei contain bothproteins (Fig. 1f–h).

Biochemical studies indicate that cyclin kinase inhibitors canform stable complexes with cyclin/CDK dimers in vitro and in

cultured cells24,27,28

. To determine whether cyclin D3 and p27Kip1

are part of a complex in Müller glial cells, we immunoprecipi-tated cyclin D3 from an adult retinal lysate. Proteins that co-

immunoprecipitated with cyclin D3 were separated bySDS–polyacrylamide gel electrophoresis (PAGE) and

immunoblotted with an antibody directed against p27Kip1. Sig-nificant amounts of p27Kip1 co-immunoprecipitated with cyclinD3 from adult retinae (Fig. 1i).

p27Kip1 is downregulated following retinal injuryTo test whether the expression levels of p27Kip1 and/or cyclin D3

changed during the proliferation response associated with reac-tive gliosis in the retina, we examined the expression of these pro-

teins following introdu ction of the neurotoxins ouabain anddomoic acid. These agents induce widespread glial cell activation

across the retina8. Adult mice received an intraocular injectionof ouabain, domoic acid or saline (PBS)8. Retinae were harvest-ed 48 hours after injection and stained with antibodies againstMüller glial cell markers. Half the retinal tissue from each eye

was dissociated for single-cell analysis, and the other half wasembedded for cryosectioning. Retinae from eyes that received an

intraocular injection of PBS showed no glial cell activation, asindicated by the very small amount of GFAP immunoreactivity inMüller glia (Fig. 2a) and the absence of detectable proliferation

(data not shown and below). In contr ast, retinae exposed toouabain or domoic acid exhibited a dramatic increase (10–20 fold)in the proportion of GFAP-immunoreactive cells (Fig. 2b and c;

Table 1). Cells upregulating GFAP appeared to downregulatep27Kip1 (Fig. 2d). When bromo-deoxyuridine (BrdU) was includ-

ed with the injected neurotoxins, GFAP immunoreactive Müllerglial cells were occasionally found to enter S-phase and incorpo-rate BrdU (Fig. 2e).

To quantify the effects of such treatment on glial cell acti-vation, we induced reactive gliosis in vitro and labeled cellswith [3H]thymidine. Adult retinae were explant cultured for

48 hours in the presence or absence of ouabain (Methods).After the first 24 hours in culture, [3H]thymidine was added

to label mitotic cells. Retinae were then dissociated, stainedwith antibodies specific for cell types or cell cycle proteins, andprocessed for autoradiography. Although the magnitude of

induction of gliosis as measured by the propor tion of GFAP-immu nor eactive cells was similar to th at ob served in vivo

(10–15 fold; Table 1), the proportion of GFAP-immunoreac-tive cells in the un treated samples was slightly higher in vitro

(0.4% as compared to 0.1% in vivo). All cells that incorporat-

ed [3H]thymidine were CRALBP-immunoreactive Müller glialcells (Fig. 2f ). We never observed [3H]thymidine-labeled rodphotoreceptors (anti-rhodopsin), bipolar interneurons (anti-

Chx10) or horizontal/amacrine cells (anti-syntaxin-1; data notshown). Significantly, all th e [ 3H]thymidine-labeled cells

expressed GFAP, as expected for reactive gliosis (Fig. 2g; Table 1).Amon g the GFAP-immu noreactive glial cells, on ly a subset

(5 ± 0.3%, 13 of 256) entered S-phase in the last 24-hour peri-od of culture in the presence of ouabain (Table 1). These dataare consistent with previous in vivo analyses8. To determ inewhether p27Kip1 downregulation correlated with the activation

of glial cell proliferation, we examined the expression of p27Kip1

in [3H]thymidine-labeled Müller glial cells (Fig. 2h). From sev-

eral independent retinae, we found no [3H]thymidine-labeledcells among 37 cells that expressed p27Kip1 (Table 1). In addi-tion, p27Kip1-immunoreactive cells, which were readily detect-

ed (Fig. 2i), never incorporated [3H]thymidine. When cyclinD3 expression was examin ed, amon g the [3H]thymidine-labeled Müller glial cells, a small number (5 of 31, 16%) were

positive (Fig. 2j) bu t a significant proportion had downregu-lated cyclin D3 (26 of 31, 84%).

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Fig. 1. Expression of p27Kip1 and cyclin D3 in theadult mouse retina. (a) Drawing of a Müller glial cellspanning all three nuclear layers of the vertebrate reti-nae. Müller microvilli make up the outer limiting mem-brane (arrow). (b–e) p27Kip1 and CRALBPimmunofluorescence in the adult retina. (b) An anti-

body specific for p27Kip1 stains a uniform row of nucleiin the inner nuclear layer whose shape and positionare consistent with Müller glial cells. (c) CRALBP isexpressed in the cytoplasm of Müller glial cells.(d) The p27Kip1 immunoreactive nuclei precisely local-ize to the CRALBP immunoreactive Müller glial cellbodies. (e) A high-magnification view of CRALBP andp27Kip1 colocalization. (f–h) p27Kip1 and cyclin D3immunofluorescence in the adult retina. (f) A high-magnification view of the p27Kip1 immunoreactivenuclei of Müller glial cells. The same field of cellsshown in (f) also exhibit cyclin D3 immunoreactivity(g), and these two proteins colocalize to the nuclei ofMüller glial cells (h). (i) Co-immunoprecipitation ofp27Kip1 with cyclin D3. Cyclin D3 was immunoprecipi-tated from crude retinal lysate. The starting material (lane 1), washes (lanes 2–4), control IgG immunoprecipitation (lane 5) and bound fractions (lane 6)

were separted by SDS–PAGE and immunoblot ted. The start ing material contained p27Kip1, and it co-immunoprecipitated with cyclin D3 (arrow).CRALBP, cellular retinaldehyde binding protein; OS, photoreceptor outer segments; olm, outer l imit ing membrane; ONL, outer nuclear layer; OPL,outer plexiform layer ; INL, inner nuclear layer ; IPL, inner plexiform layer; GCL, ganglion cell layer; Mr, protein markers, relative molecular mass frombottom, 26, 36, 42, 66, 97, 116, 158 kDa. Scale bars, 100 µm (b–d), 10 µm (e–h).


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To delineate the order of the changes in p27Kip1, GFAP and

cyclin D3 expression in proliferating Müller glial cells dur ingreactive gliosis, we did a [3H]thymidine pulse-labeling experi-ment . Retinae were cultured for 24 hou rs in the presence of

ouabain, and then labeled for 2 hours with [3H]thymidine tomark cells in S-phase. Tissue was then either harvested immedi-

ately or following a culture period of 4, 8, 16, 24 or 48 hours in

the absence of [3H]thymidine. Immediately after [3H]thymidinelabeling, all [3H]thymidine-labeled Müller glial cells lackedexpression of p27Kip1 and m aintained expression of cyclin D3

(Table 1). Few of these cells (1 of 29, 2%) had upregulated GFAP(Fig. 2k). By 4 hours after labeling, GFAP immunoreactivity was

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Table 1. Analysis of protein expression in proliferating Müller glial cells following ret inal injury.

Treatment CRALBP+ /total GFAP+ /total GFAP+,[3H]thy+ /GFAP+ p27Kip1+,[3H]thy+ /[3H]thy+

(counts; mean %± s.d.)1 (counts; mean %± s.d.) (counts; mean %±s .d.)2 (counts; mean %± s.d.)3

Ouabain 32/500, 44/500, 36/500, 21/500, 24/500, 21/500, n.a.4 n.a.(in vivo) 47/500 18/500

(7.9 ± 1.4) (4.2 ± 0.5)

PBS 27/500, 34/500, 28/500, 1/500, 1/500, 0/500, n.a. n.a.(in vivo) 26/500 0/500

(5.7 ± 0.7) (0.1 ± 0.1)

Ouabain 23/500, 29/500, 31/500 35/500, 37/500, 38/500 8/147, 5/109 0/20, 0/17(in vitro) (5.5 ± 0.8) (7.2 ± 0.4) (5.2 ± 0.3) (0)

PBS 22/500, 19/500, 35/500 3/500, 4/500, 2/500 0/9, 0/11 n.a.5

(in vitro) (5.1 ± 1.7) (0.6 ± 0.2) (0)

1Adult (6 week old) mice received a 0.5–1.0 microliter injection of ouabain in the left eye and PBSin the right eye. Forty-eight hours after injection, retinaewere harvested, dispersed, plated and stained by immunohistochemistr y. For in vitro analysis, freshly dissected retinae were explant cultured in the presenceor absence of ouabain for 48 hours. Four independent animals were analyzed in vivo , and three animals were analyzed in vitro . 2Due to the relative scarcity ofGFAP-immunoreactive cells, the fields of dispersed cells were searched for GFAP immunoreactivity and then scored with respect to [ 3H]thymidine incorpora-tion. Therefore, the total number of cells scored is not presented but can be inferred from the total proportion of GFAP-immunoreactive cells. The number

of silver grains in labeled cells varied from 23 to 65 (mean, 34 ± 17) and the number of silver grains in unlabeled cells ranged from 0 to 6 (mean, 2 ± 1.4). 3Dueto the sparse distribution of [3H]thymidine-labeled cells, the fields of dispersed cells were searched for [3H]thymidine labeling and then scored for p27Kip1

immunoreactivity. Therefore, the total number of cells scored is not presented. 4Data on the [3H]thymidine labeling of cells from in vivo treatment is not avail-able because [3H]thymidine was not injected into the eyes of adult animals. 5Data on the [3H]thymidine labeling of cells from in vitro treatment with PBSis notavailable because adult retinal cells do not proliferate.

Fig. 2. Changes in gene expression in Müller glial cells following reti-nal injury. (a–e) Analysis of GFAP expression, p27Kip1 expression, and

BrdU incorporation following injection of neurotoxins. (a) Forty-eight hours after an intraocular injection of 0.5 microliters of PBS,GFAP expression was only detected in the rare astrocytes (As) alongthe vitreal surface of the retina. (b) GFAP immunoreactivity wasobserved in Müller glial cells (Mü) after neuronal toxicity brought onby intraocular injection of ouabain or domoic acid (not shown). ( c) Ahigh-magnification view of a GFAP-immunoreactive Müller glial cell.(d) Immunohistochemical staining with antibodies directed againstGFAP (red fluorescence) and p27Kip1 (arrow, green fluorescence)indicated that the Müller glial cells that expressed GFAP had down-regulated p27Kip1. (e) W hen BrdU was injected along with ouabain,ocassionally a GFAP immunoreactive (red fluorescence) Müller cellhad entered S-phase as measured by BrdU immunofluorescence(arrow, green fluorescence). (f–i) Analysis of the gene expressionkinetics in explanted retinae treated with neurotoxins. Gene expres-sion was assayed by single-cell immunofluorescence (FITC).

Proliferation was measured by the uptake of [3

H]thymidine (br ightfield) in the nuclei (DAPI) of individual cells expressing a given protein.(f) CRALBP immunoreactive Müller glial cells re-entered the cell cycleafter treatment with ouabain. (g) Müller glial cells that re-entered thecell cycle upregulated GFAP. Müller glial cells that re-entered the cellcycle never expressed p27Kip1 (h), and those that continued toexpress p27Kip1 were never found to incorporate [3H]thymidine (i).(j) Forty-eight hours after treatment with ouabain, a small fraction ofmitot ic Müller glial cells expressed cyclin D3. (k) Kinetics of cyclin D3and GFAP expression following S-phase entr y. Immediately after pulselabeling with [3H]thymidine, most of the cells that incorporated[3H]thymidine expressed cyclin D3 and showed no GFAP expression.Several hours later, the fraction of cells expressing cyclin D3 wasreduced, whereas the propor tion of cells expressing GFAP increased.PBS, phosphate buffered saline; GFAP, glial fibr illary acidic protein; INL, inner nuclear layer ; ONL, outer nuclear layer; GCL, ganglion cell layer;CRALBP, cellular retinaldehyde binding protein; BrdU, bromo-deoxyur idine. Scale bars, 100µm (a–d), 10 µm (e–i).

Time after labeling ( hours)

P e r c e n t [ 3 H ] + ,

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detected in significantly more [3H]thymidine-labeled Müller glialcells (14 of 30,47%), and this propor tion reached a maximum24 hours after labeling (Fig. 2k). During this same time interval,

cyclin D3 was downregulated in Müller glia that were in S-phaseat the time of labeling (Fig. 2k).

Reactive gliosis in the retinae of p27Kip1-deficient miceTo determine if loss of p27Kip1 expression was sufficient for Müller

glial cell induction, we examined retinae from p27Kip1-deficientmice25,29,30. Retinae from adult p27Kip1–/– mice and their het-

erozygous or wild-type littermates were dispersed or cryosec-tioned and used for immunohistochemical studies.GFAP-immunoreactive glial cells were found throughout the reti-

nae of p27Kip1-deficient mice (Fig. 3a), but not in their wild-typeor heterozygous littermates (Fig. 3b; data not shown). Quanti-tative analysis of dispersed retinal cells revealed a 10–20-fold

induction of GFAP immunoreactive cells with distinctive Müllerglial cell morphology (Fig. 3c and d). To determine whether cyclin

D3 was downregulated in the retinae from p27Kip1-deficient mice,we did immunohistochemical staining using an antibody direct-

ed against cyclin D3. Müller glial cells from wild-type andp27Kip1+/– retinae expressed cyclin D3 (Fig. 3e and f ), whereas reti-nae from the p27Kip1-deficient mice showed no detectable cyclinD3 expression (Fig. 3g). These results are similar to the data

obtained on retinae treated with neurotoxins (above).

Reactive gliosis leads to retinal dysplasiaOne of the hallmarks of retinae from p27Kip1-deficient mice isdysplasia (Fig. 4a–d). The presence of p27Kip1 and cyclin D3 in

Müller glial cells, combined with the suggestion that the reti-nae from mice lacking p27Kip1 have defects in their outer limit-ing membranes25, led us to test whether inappropriate Müller

glial cell activation during development could have caused theretinal dysplasia of p27Kip1-deficient mice. As a first step toward

testing this hypothesis, we examined the retinal dysplasia ingreater detail using antibodies to specific retinal cell typesand/or structures.

CD44 is expressed at high levels in the apical microvilli of Müller glial cells, which make up the outer limiting membrane

of the retina31

. Using an antibody directed against CD44, wefound the outer limiting membrane to be disrupted in retinaefrom p27Kip1-deficient mice at the sites of retinal dysplasia (Fig. 4e

and i). In each region where the outer limiting membrane wasdisrupted, the processes of activated (GFAP immunoreactive)

Müller glial cells were present and, in many cases, extendedbeyond the boundary of the outer limiting membrane (Fig. 4f

and j). By using ant ibodies directed against a variety of cell-type-

specific markers, we found that most of the cells that made upthe retinal dysplasia were rhodopsin-immunoreactive rod pho-toreceptors (Fig. 4g and k). Occasionally, a calbindin/neurofila-

ment-immunoreactive horizontal cell was found outside the outerlimiting membrane (Fig. 4h and l), displaced from its normal

position at the INL/ONL boundary.To test the possibility that retinal dysplasia was the result of

ectopic proliferation as proposed25, we did in vivo lineage analysison the knockout retinae. If retinal dysplasia resulted from dereg-ulated proliferation , we would expect to see abnormally largeclones associated with the dysplastic lesions in p27Kip1–/– retinae.

Newborn mouse pups from a mating of p27Kip1+/– animals receivedintraocular injections of the replication-incompetent retrovirus

LIA encoding alkaline phosphatase32. Following complete retinaldevelopment (3 weeks), retinae were harvested, stained for alka-line phosphatase expression and sectioned, and clones of cells

derived from single infected progenitor cells were scored for cellnumber and cell type. In the p27Kip1-deficient retinae, 17 cloneswere found within or immediately adjacent to regions where reti-

nal dysplasia was apparent. Most of these were single-cell (10 of 17; Fig. 4m) or two-cell (6 of 17; Fig. 4n) clones; none of them

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Fig. 3. Characterization of Müller glial cells in the retinae of p27Kip1-deficient mice. (a, b) GFAP immunoflurescence in p27 –/– and p27+/– reti-nae. Retinae were harvested from six-week-old mice lacking the p27Kip1

gene or their heterozygous littermates, and cryosections were incu-bated with an antibody specific for GFAP. (a) Extensive GFAPimmunoreactivity was observed in Müller glial cells of the p27Kip1-defi-

cient retinae. (b) Heterozygous and wild-type (data not shown) litter-mates had no GFAP immunoreactivity beyond the astrocyte stainingobserved normally along the vit real surface. (c, d) Single-cell staining forMüller glial cell markers. (c) A single GFAP-immunoreactive Müller glialcell from a p27Kip1-deficient retina, which maintained its distinctive mor-phology even after the tissue was dissociated. (d) Retinae from p27Kip1-deficient, heterozygous or wild-type littermates were dispersed andplated, and individual cells were examined for the expression ofCRALBP or GFAP. The propor tion of Müller glial cells was similar inmice lacking p27Kip1 as measured by CRALBP immunoreactivity.However, the propor tion of GFAP-immunoreactive Müller glial cells wassubstantially increased in the p27Kip1-deficient retinae. Each bar repre-sents the average of 1000 cells scored for 2–4 independent retinae.*Some samples had so few GFAP immunoreactive cells (0–1) that thebars are not visible on the graph. (e–g) Cylin D3 expression in the reti-nae of p27Kip1-deficient mice. (e) Retinae from wild-type mice show the

normal pattern of Müller glial nuclear immunoreactivity.(f) Heterozygous littermates exhibited a slight disorganization of theMüller glial cell layer, but cyclin D3 expression persisted. (g) Müller glialcells in the retinae from mice lacking p27Kip1 lacked cyclin D3 expres-sion. GFAP, glial fibr illary acidic prot ein; CRALBP, cellular retinaldehydebinding prot ein; ONL, outer nuclear layer ; INL, inner nuclear layer;GCL, ganglion cell layer. Scale bars, 100 µm (a, b, e–g), 10 µm (c).

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contained more than three cells. Approximately 100 clones foreach group (p27Kip1–/–, p27Kip1+/–, p27Kip1+/+) were found through-out the retina (data not shown). The average size and composi-

tion of clones from mice lacking one or both alleles of p27Kip1 weresimilar to those identified in their wild-type littermates (data not

shown) and was consistent with previous data33,34.To test whether the induction of reactive gliosis during devel-

opment of wild-type animals can lead to retinal dysplasia, and

whether there is a correlation with an alteration in the outer lim-iting membrane, we administered an intraocular dose of ouabainto mouse pups at postnatal day 10.5. This is the stage when

Müller glial cells are differentiating and retinal dysplasia is first

seen in the p27Kip1

-deficient retinae (M.A.D. and C.L.C., unpub-lished data). Two weeks after the injection, when retinal devel-opment was complete, retinae were harvested, sectioned and

stained with antibodies to GFAP, p27Kip1, cyclin D3 and the outerlimiting membrane marker, CD44. We observed retinal dysplasia

in the retinae treated with ouabain that was similar to that foundin p27Kip1-deficient mice (Fig. 5). Retinae from eyes that receivedan intra-ocular injection of saline never showed retinal dyspla-

sia (data not shown). In the dysplastic regions of ouabain-inject-ed eyes, CD44 immunoreactivity was absent, indicatingdisruption of the outer limiting membrane (Fig. 5a and e). Fur-

thermore, Müller cells initiated a program of reactive gliosis at

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Fig. 4. Characterization ofthe retinal dysplasia foundin p27Kip1-deficient mice.(a–d) Retinal dysplasia inp27Kip1-deficient retinae. DAPIstaining (a) and DIC

microscopy (b) reveal cellbodies displaced beyond theouter limiting membrane intothe region normally occupiedby photoreceptor outer seg-ments. (c, d) DAPI stainingand DIC microscopy of aretina from the wild-type lit-termate to the p27Kip1-defi-cient animal shown above.(e–l) Detailed characteriza-tion of retinal dysplasia foundin p27Kip1-deficient mice.Photographs (e–h) and trac-ings (i–l) of immunostainingusing antibodies to specific

retinal structures or cell types.(e, i) The outer limiting mem-brane was disrupted in regionsof retinal dysplasia (arrow) asindicated by an alteration inthe CD44 immunoreactivity. (f, j) GFAP-immunoreactive Müller glial cell processes were found wherever the outer limit ing membrane was disruptedand retinal dysplasia formed. (g, k) Most cells that breeched the outer limiting membrane were rhodopsin-immunoreactive photoreceptors.(h, l) Occasionally a neurofilament/calbindin-immunoreactive horizontal cell was found displaced from its normal position at the ONL/INL boundary to theouter segment layer. (m, n) In vivo lineage analysis in p27Kip1-deficient retinae. Replication-incompetent retroviruses were injected into the eyes of newbornmice from a cross of p27Kip1 heterozygous parents. (m) A single alkaline-phosphatase-expressing cell in the region of retinal dysplasia (arrows) of the p27Kip1-deficient mouse. (n) An example of a two-cell clone containing a bipolar interneuron in the INL. Dashed lines indicate the boundary between the ONL andphotoreceptor outer segments. DIC, differential interference contrast; OS, photoreceptor outer segments; OLM, outer limiting membrane; ONL, outernuclear layer; INL, inner nuclear layer; GFAP, glial fibrillary acidic protein; Rho4D2, anti-rhodopsin antibody. Scale bars, 50µm (a–d, m,n), 20µm (e–l).

Fig. 5. Induction of reactive gliosis during reti-nal development. Intraocular injection of

oubain at postnatal day 10.5 led to retinal dys-plasia (arrows delineate the boundaries) simi-lar to that found in the retinae fromp27Kip1-deficient mice. (a–d) Photographs ofimmunostaining using antibodies to specificretinal structures or Müller glial cell markers inthe regions of retinal dysplasia (e–h). (a) Adecrease in CD44 immunofluorescence indi-cates disruption of the outer limiting mem-brane at the site of retinal dysplasia followingintra-ocular injection of ouabain at P10.5.(b) GFAP immunofluorescence was also con-centrated at the sites of retinal dysplasia.(c, d) Both p27Kip1 (c) and cyclin D3 (d) weredownregulated in the nuclei of Müller cells undergoing reactive gliosis (open arrowheads). olm, outer limit ing membrane; ON L, outer nuclear layer;INL, inner nuclear layer; GCL, ganglion cell layer; GFAP, glial fibr illary acidic protein. Scale bars, 100µm.


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the sites of retinal dysplasia; GFAP was upregulated (Fig. 5b andf ), p27Kip1 was downregulated (Fig. 5c and g), and cyclin D3 wasdownregulated (Fig. 5d and h). These molecular events precise-

ly mimic those observed in p27Kip1-deficient retinae and in adultretinae treated with neurotoxins.

Vascular defects associated with reactive gliosisPrevious work on human retinopathies associated with diabetes

and on animal models suggested that Müller glial cells are involvedin the very earliest steps of diabetic retinopathy12,13,35,36. To test

whether glial cell activation might lead to alterations in retinal vas-culature, we did immunohistochemical staining using an antibodydirected against the vascular endothelial cell marker, PECAM-1.

Normally, the retinal vasculature is found in the plexiform layers,the GCL and INL, and along the vitreal surface of the retina. Trans-verse sections through these structures generally give cross sections

of the retinal vasculature with branched vessels sometimes appear-ing parallel to the plane of the section in the inner nuclear layer

(INL;Fig. 6a–c). Two major alterations in the vascular structures of wild-type retinae with reactive gliosis or p27Kip1-deficient retinae

were observed. First, more extensive INL vascularization was seenin regions of retinal dysplasia and reactive gliosis (Fig. 6d–g). Sec-ond, an enlargement of the vessels was observed in retinae fromp27Kip1-deficient mice, and blood cells were often seen outside of

these structures (Fig. 6h and i and data not shown).

DISCUSSION

Nearly every major disease of the retina, including retinitis pig-mentosa14,37,38, macular degeneration39–42 and diabetic retinopa-

thy13,35,36, is associated with reactive gliosis involving Müllerglial cells. Despite the importance of this association, no genesthat regulate this process have been reported. Our experiments

demonstrate that the level of p27Kip1 expression in Müller glialcells is critical for regulation of reactive gliosis. Following acute

retinal injury, Müller glial cells rapidly downregulated p27Kip1

protein levels and re-entered the cell cycle. Shortly thereafter,the levels of the inter mediate filament protein, GFAP, were

increased. Subsequently, additional rounds of cell division werenot observed, most likely because of the observed downregu-

lation o f cyclin D3 protein in the activated Müller glial cells.The causal role of p27Kip1 in reactive gliosis was demonstrated byobservations on p27Kip1-deficient mice. Retinae from mice lack-

ing p27Kip1 exhibited reactive gliosis throughout the retina. Wepropose th at th e retinal dysplasia reported p reviously in

p27Kip1–/– animals results from disruptions in the outer limitingmembrane due to reactive gliosis during development. We alsofound alterations in the vasculature of retinae from p27Kip1-defi-

cient mice or retinae of wild-type mice treated with excitatoryamino acids. Taken together, our experiments indicate that theregulation of Müller glial cell proliferation occurs via p27Kip1,

and that cyclin D3 regulation ultimately contributes to keep-ing Müller cell proliferation in check. In addition, these data

show the crucial role of Müller cells in the formation of retinaldysplasia and support the notion that Müller cells are key reg-

ulators of retinal vasculature.We found that p27Kip1 downregulation is the earliest indicator

of Müller glial cell activation identified to date. GFAP is upregulat-ed quickly in Müller glial cells following retinal injury8,9. Here we

report that p27Kip1 downregulation preceeds GFAP upregulation.Why might p27Kip1 regulate both proliferation and GFAP levels,

with proliferation preceeding GFAP upregulation? Intermediate fil-aments are broken down and reassembled when cells undergo mito-sis43. It is possible that Müller glial cells coordinate mitosis with

GFAP upregulation to efficiently incorporate these molecules intothe cytoskeleton during reassembly following cytokinesis, perhapsexplaining why cell cycle re-entry precedes GFAP upregulation.

Although proliferation occurs during reactive gliosis, glialtumors are not common, implying that some mechanism(s) to

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Fig. 6. Examination of the vasculature in retinae under-going reactive gliosis. (a–c) Normal vascular structure inthe adult retina. (a) Transverse section of a normal retinastained with an anti-PECAM-1 antibody. Vascularendothelial structures are indicated by arrows. (b) Thesevessels are found primarily in the INL, the plexiform lay-

ers, and adjacent to the ganglion cell layer of the verte-brate retina as shown by this DIC image of the sameretinal section shown in (a). (c) Whole-mount stainingfor PECAM-1 in a normal adult retina photographedfrom the vitreal side of the tissue. The optic nerve isapproximately 1 mm to the left. (d–f) PECAM-1 stainingon retinae following an intra-ocular injection of ouabainat P10.5. (d) PECAM-1 immunoreactivity was moreextensive in these retinae. (e) DIC image of the retinalsection shown in (d). (f) Whole-mount staining verifiedthe increases in retinal vasculature. As in (c), the opticnerve lies approximately 1 mm to the left. (g–i) PECAM-1staining on retinae of p27Kip1-deficient mice. (g) The reti-nae from six-week-old p27Kip1-deficient mice showedextensive vasodialation and neovascularization. Theouter nuclear layer is not visible in this photograph.

(h) High-magnification view of a particularly largePECAM-1 immunoreactive vessel, which contained largenumbers of red blood cells (i) within the vessel and in thesurrounding tissue (arrows). ONL, outer nuclear layer;opl, outer plexiform layer; ipl, inner plexiform layer; INL,inner nuclear layer; GCL, ganglion cell layer. Scale bars,100µm (a, b, d, e, g, h), 0.5 mm (c, f), 10µm (i).


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limit proliferation must be present. We found that cyclin D3was downregulated approximately 16–24 hours after Müller

glial cells initiated reactive gliosis. Without an interacting cyclin,the cyclin-dependent kinase(s) in Müller glial cells would beunable to phosphorylate the retinoblastoma protein (or its relat-

ed family members), and Müller glial cells would enter a qui-escent G0 state24. It is intriguing that cyclin D3 wasdownregulated following cell division rather than p27Kip1 lev-

els returning to normal. Presumably, the signals from damagedneurons that led to Müller glial cell activation and p27Kip1 down-

regulation in th e first place were still present after the first48 hours in culture. Therefore, perhaps to prevent further celldivision, cyclin D3 was downregulated.

If p27Kip1 levels determine the entry of Müller cells into reac-tive gliosis, as opposed to simply being correlated with it, thenthe p27Kip1-deficient retinae should exhibit reactive gliosis with-

out induction by any of the known stimulants of reactive gliosis.This was indeed the case. At postnatal day 10.5 when Müller glial

cells are normally differentiating, we observed inappropriate S-phase entry and apoptosis in p27Kip1-deficient retinae (M.A.D.and C.L.C., unp ublished data). Notably, GFAP expression in

p27Kip1–/– Müller glial cells was detected as early as P10.5 and per-sisted into the adu lt stages44 (M.A.D. and C.L.C., unpublisheddata). However, proliferation ceased shortly after Müller glia were

formed, in concert with the downregulation of cyclin D3 in theMüller glial cells of the p27Kip1-deficient mice (M.A.D. and C.L.C.,

unpublished data). We did not observe an increase in the totalnumber of Müller glial cells as measured by CRALBP immunore-activity in the adult retina (Fig. 3d), which may reflect limited

proliferation or elimination by apoptosis in the p27Kip1-deficientretina (M.A.D. and C.L.C., unpublished data). Overall, the resultsfrom p27Kip1-deficient mice are consistent with a model in which

p27Kip1 levels control the initiation of reactive gliosis in the retina,and, most likely, cyclin D3 downregulation prevents uncontrolled

Müller cell proliferation.In the p27Kip1–/– retina, dysplasia does not seem to result

from photoreceptor proliferation as previously reported25. The

hypoth esis of photo receptor pro liferation was based onimmunohistochemical staining using an antibody specific for

p27Kip1, which demonstrated immunoreactivity in the outersegments of photoreceptors25. Restriction to outer segments isnot consistent with the identification of a p utative nuclear

localization signal near the carb oxy termin us of p27Kip1

(ref. 45), and we could find no evidence for p27Kip1 expressionin the nuclei or outer segments of photoreceptors. Therefore, it

is likely that the previously reported expression of p27Kip1 inphotoreceptor outer segments was the result of non- specific

antibody binding to the outer segments, which is a commonartifact of retinal immunostaining. Beyond this disparity in

the expression pattern, in vivo lineage analysis, BrdU labeling of adult retinae, and examination of dysplastic regions in olderanimals argue against deregulated photoreceptor cell prolifer-ation leading to the observed retinal dysplasia in p27Kip1-defi-

cient mice. Instead, we found that when reactive gliosis wasinduced by administration of an intraocular injection of

ouabain before the completion of retinal maturation (P10.5),we observed retinal dysplasia that was very similar to that seenin retinae from p27Kip1-deficient mice. It is likely that the

changes in the shape and structure of Müller glial cells associ-ated with alterations in intermediate filament compon ents(GFAP) led to a disruption in the outer limiting membrane

and subsequent dysplasia in that region. Furthermore, thesedata indicate that the structural integrity of the mature retina

relies on an intact outer limiting membrane during develop-ment. Perturbations in this structure result in retinal dyspla-

sia and a significant reduction in phototransduction 25.Müller glial cells undergoing reactive gliosis are believed to

alter the local environment for neurons and to provide additional

structural integrity to the retina at the site of injury by the afore-mentioned changes in intermediate filament constituents. How-ever, when reactive gliosis is deregulated to the point where it

results in so-called ‘massive gliosis,’ or under conditions of chron-ic Müller glial cell activation such as ischemia associated with

diabetes, severe neuronal damage may lead to blindness. In thecase of diabetic retinopathy, the prolonged secretion of vascularendothelial growth factor, a potent vasodialator and vascular

endothelial mitogen, by Müller glial cells could contribute to theprogression of this debilitating disease12,13. Evidence from theretinae of p27Kip1-deficient mice and injured retinae lend sup-

port to this hypothesis. Our initial characterization suggests thatMüller glial cell activation results in vasodilation and vessel

growth or reorganization. These results demonstrate that Müllerglial cell activation per se (targeted disruption of p27Kip1 and exci-tatoxicity) can lead to vascular changes in the vertebrate retina.

These findings may be of considerable clinical relevance if thesame molecules are found to regulate Müller glial cell prolifera-tion and reactive gliosis in the human retina.

M ETHODS

Animals. C57BL/6 and ICR mice were purchased from Taconic Farms

(Germantown, New York). The p27Kip1-deficient mice30 were crossed to

ICR or C57BL/6 mice with equivalent results. Genotypes were deter-

mined by PCR amplification of the wild-type and mutant alleles from

tail DNA30.

Immunohistochemistry and antibodies. Immun ohistochemical stain-

ing of retinal cryosections or dissociated cells was done as described46.

Many of the ant ibodies used for th ese stud ies have been described46

.Other antibodies were anti-CD31 (PECAM-1), MEC 13.3 (rat mono-

clonal, 1:100; Pharmingen) and anti-CD44, 5D2-27(rat monoclonal,

1:100; Developmental Studies Hybridoma Bank). Detailed protocols can

also be found at http://axon.med.harvard.edu/ ∼cepko/protocol/mike/.

[3H] thymidine labeling, retinal explant cultures and dissociation. To

label retinal progenitor cells in S-phase, we incubated retinae in 1 ml

explant culture medium46 containing [3H] thymidine (NEN, 5 µCi/ml;

89 Ci/mmol) for 1 hour at 37°C. Autoradiography was done as

described47. The procedure for explant culturing of retinae has been

described46 and is available at http://axon.med.harvard.edu/ ∼cepko/pro-

tocol/mike/. Extensive characterization h as demonstrated that retinal

proliferation and differentiation are normal using this explant culture

system46. Tissue dissociation was done as described47.

Replication-incompetent retroviral vector constructs and viral pro-duction. To prepare high-titer retroviral stocks, the plasmid construct

pLIA-E (M.A.D. & C.L.C., unpublished data) , which is similar to pLIA48,

was transiently transfected into a 293T ecotropic produ cer cell line

(Phoenix-E) by calcium p hosphate co-precipitation as described48 .

Supernatant containing the viral particles was harvested at 48 hours after

transfection, and viral titer was determined on NIH-3T3 cells48. In vivo

lineage analysis was done as described33,34.

Co-immunoprecipitation and immunoblotting. For cyclin D3 co-

immun oprecipitation, 10 adult (3 week) mouse retinae were briefly son-

icated in 2 ml RIPA buffer (PBS, 1% NP-40, 0.5% sodium deoxycholate,

0.1% SDS) con taining 1 mM PMSF, a cocktail of protease inhibitors

(Sigma), and phosphatase inhibitors (1 mM Levamisole, 2 mM Na2VO3,

1 mM NaF). Anti-cyclin D3 (C-16, rabbit polyclonal, Santa Cruz Biotech,

Santa Cru z, California) ant ibody was incubated with the crude retinal

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lysate and immunoprecipitated with protein-G agarose according to the

manu facturer’s instru ctions ( Santa Cruz Biotech). SDS–PAGE and

immunoblotting were done as described46.

Induction of reactive gliosis. For in vitro induction of reactive gliosis,

retinae from mature (3 week old) mice (c57BL/6) were dissected away

from the surrounding tissue, and the lens was removed. Only retinaelacking holes or tears, which could result in reactive gliosis, were used

for this procedure. Retinae were then explant cultured 46 in the pres-

ence of domoic acid (Sigma, 20 µM) or ouabain (Sigma, 70 µM) for

48 hours49. For analysis of mitotic cells, [3H]thymidine (5 µCi/ml) was

added after the initial 24 hours in culture. Following this culture peri-

od, retin ae were processed for cryosectioning, dissociation an d/o r

autoradiography as described above. For in vivo induction of reactive

gliosis, 0.5–1.0 microliters of neurotoxins (domoic acid 2 mM, ouabain

7 mM) were injected into the eyes of 3–6 week-old C57BL/6 mice anes-

thetized by ether inhalation as described49. For labeling of mitotic cells

in vivo, BrdU ( 1 mM) was injected along with the aforement ioned

compounds. Retinae were harvested 48 hours after injection. Induc-

tion of reactive gliosis in P10.5 retinae was achieved by injecting 0.5–1.0

microliters of ouabain at 0.7 mM. Similar results were obtained from

retinae exposed to neur otoxins in vivo and in vitro; however, back-

ground induction of reactive gliosis was slightly higher in vitro due topunctures or microtears.

ACKNOWLEDGEMENTS

We thank M .H. Baron for discussion and support throughout this project,

S. Elledge, W. Harper and P. Zhang for cDNAs; J. Roberts and L.H. Tsai for

knockout mice, and J. Zitz, M. Peters and L. Rose for technical support.

M.A. Dyer was supported by NRSA fellowship # EY06803-02 and the

Charles H. Revson Foundation Fellowship for Biomedical Research. This work

was supported by National Institutes of Health Grant # EY0-8064.

RECEIVED 26 JUNE;ACCEPTED 28 JULY 2000

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