Andy J. Fischer and Thomas A. Reh- Identification of a Proliferating Marginal Zone of Retinal Progenitors in Postnatal Chickens

8/3/2019 Andy J. Fischer and Thomas A. Reh- Identification of a Proliferating Marginal Zone of Retinal Progenitors in Postnata

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Identification of a Proliferating Marginal Zoneof Retinal Progenitors in Postnatal Chickens

Andy J. Fischer and Thomas A. Reh1

Departm ent of Biological Structure, University of Washington, Seattle, Washington 98195

n warm-blooded vertebrates it is generally accepted that after early s tages of developmen t new neurons are not added to the

etina. Con trary to this belief, we show here th at hatched chi ckens have a zone of proliferating cells at the peripheral m argin

f the retina, sim ilar to t hat of fish and amphibians. We found that cells at the peripheral edge of the retina incorporated the

hym idine analog BrdU and expressed the cell cycle regulator proliferating cel l nucle ar antigen (PCNA). Furthermore, cells

n the ciliary epithelium and retinal margin coexpressed the homeodomain transcription factors Pax6 and Chx-10, similaro multipotent progenitors of embryonic retina. Expression of PCNA, Pax6, and Chx-10 in cells at the retinal margin was

maintained in adult birds. Double-labeling studies showed that BrdU-labeled cells that were integrated into the retina

xpressed proteins found only in differentiated neurons. Increased rates of ocular growth, induced by visual deprivation,

esulted in increased numbers of BrdU-labeled cells at the retinal margin. Unlike the progenitors in the retinal marginal

one o f fish and am phibians, th e progenitors of the c hick retina do no t in crease thei r rate of proliferation in response to acute

amage. Furthermore, insulin, insulin-like growth factor-I, and epidermal growth factor increased proliferation of

rogenitors at the retinal margin, while basic fibroblast growth factor had no effect. These results indicate that the avian

etina has a marginal growth zone containing proliferating cells that share similarities with multipotent embryonic retinal

rogenitors and the retinal stem cells of cold-blooded vertebrates. 2000 Academic Press

Key Words: retina; chick; neurogenesis; progenitors; form-deprivation myopia; Chx-10; Pax6; BrdU; immunocytochemistry.

NTRODUCTION

Retinal histogenesis in warm-blooded vertebrates (birds

nd m amm als) is th ought to occur only during early stages

f development. For example, all cell types in the chick

etina are believed to be formed more than 1 week before

atch ing (Prada et al., 1991). Differentiation of cells begins

round Embryonic Da y 2 (E2) in the c entra l re tina a nd

rogresses to peripheral regions. The first cells formed

uring retinal neurogenesis are the ganglion cells (Kahn,

973), followed closely by photoreceptors, amacrine and

oriz onta l c ells (Fujita a nd Horri , 1963; M orris, 1973;

pence and Robson, 1989; Prada et al., 1991). Th e last cell

ypes formed are th e bipolar cells and M uller glia, with th e

nal cell divisions in the retina reported to occur around

12 (Kahn, 1974; Prada et al., 1991). The chick retina is

ully functional and believed to be postm itotic at t he t ime

f ha tc hing, a bout E21. Howe ve r, M orris e t a l. (1976)

rovided a brief accoun t th at [H 3]thym idine accum ulates in

some cells at th e extreme periphery of the postnatal chick

retina . Us ing m ode rn te c hnique s a nd re age nts , he re we

tested whether neurogenesis continues at the retinal mar-

gin of hatched chicks.

During postnatal development, the eye continues to grow

in parallel wit h overall body size, and m ost vertebrates,

including humans, are born hyperopic with their eyes too

sm all relative to t he refractive power of the lens and cornea

(Cu rtin, 1985). Postnatally, wh en t he eyes first receive clear

images, rates of ocular growth are increased, regulated by

retinal processing of visual information, so that eye size is

precisely m atched to refractive power (Wallman, 1993).

Ocular shape and size are determ ined by the growth of the

sclera, th e out er connective tissue sheath of the eye.

In a m p h ib ia n s a n d t e le os t fi s h , g ro w t h o f t h e e ye i s

coordinated with the addition of new neurons at the periph-

eral margin of the retin a and, in t he case of teleosts, new rod

photoreceptors in central regions (Johns and Fernald, 1981

Johns, 1982). In some cold-blooded vertebrates, new neu-

rons at the retinal m argin are generated by a population of

stem cells that persist throughout life (reviewed by Hitch-

cock and Raymond, 1993; Raymond and H itchcock, 1997

1 To whom correspondence should be addressed. Fax: (206) 543-

524. E-m ail: tom [email protected] ington.edu.

evelopment al Biology 220, 197210 (2000)

oi:10.1006/dbio.2000.9640, available online at htt p://www .idealibrary.com on

012-1606/00 $35.00

opyright 2000 by Academic Press

ll rights of reproduction in any form reserved. 19 7


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eh and Levine, 1998; Perron et al., 1998). In birds and

mammals, however, postnatal ocular growth is n ot believed

o be coincident with the addition of new n eurons at either

he margin or the central regions of the retina. Instead, a

assive stretching of retina is thought to occur t o compen-

ate for eye growth (Teakle et al., 1993). Here we provide

vidence that in postnatal chicks there is a proliferating

m arginal zone containing cells that share similarities withmbryonic retinal progenitors and t he retinal stem cells of

s h a n d a m p h ib ia n s. T h i s z on e p ro du c es n e w n e u ro n s

uring a dole sc ent ocula r growth a nd is pre se nt in a dult

nimals. We also show that the proliferation of progenitors

t the retinal margin can be increased by enhancing rates of

cular growth or by applying a variety of growth factors.

METHOD S A N D MATERIALS

Animals. T h e u s e o f a n i m a ls i n t h e se e xp er im e n t s w a s i n

ccordance with the guidelines established by the National Insti-

ut es of Health and the University of Washington. Newly h atchedeghorn chickens (Gallus gallus dom esticus) were obtained from

&N Highline International (Seattle, WA) and k ept on a cycle of

6 h light, 8 h dark (lights on at 6:00 am). Chicks were housed in

lear N algene cages at about 25C and received wat er and Purina

hick starter ad libitum .

Injections. Intraocular injections of BrdU were used to label

ividing cells in the retina. Chicks were anesthetized with halo-

hane pr ior to injection. I njections wer e made into the vitr eous

hamber using a 25-l Hamilton syringe with a 26-gauge needle.

enetr ation of the needle was made consistently into the dorsal

alf of the eye. The left eye (control) was injected with 20 l of

erile saline and the right eye (treated) was injected with BrdU

etween 0.5 and 10 g) dissolved in sterile saline. To test whetherr owth factor s influence the proliferation of cells at the r et inal

mar gin we injected eyes at P2 and P 3 with Br dU (0.5 g) and

ifferent growth factors, and harvested eyes at P7 to process them

or BrdU immunoreactivity. Growth factors used in these experi-

ments included pur ified bovine insulin (500 ng), r ecombinant

u m an in sulin-like growth factor-I (IGF-I, between 1 and 100 n g per

njection), recombinan t h um an epiderm al growt h factor (EGF, 10 or

00 ng per injection), and purified bovine basic fibroblast growth

actor (bFGF, 10 or 100 ng per injection). All growth factors were

btained from R & D Systems and were dissolved in saline plus 0.1

m g/ml BSA and 0.5 g BrdU. In anoth er set of experiment s, a toxic

ose of N-methyl-D-aspartate (NM DA; 280 g) was injected into

he left eye of newly hatched chicks, followed by four consecutive

aily injections of 0.5 g of BrdU. All drugs w ere obtained fromigma.

Visual deprivation experiments. Newly hatched chicks wer e

orm-deprived by gluing a translucent light-diffusing goggle over

heir left eye. In six chicks, injections of BrdU (1.0 g/dose) were

made into the vitreous chamber (as described above) of both eyes

or 4 consecutive days. On t he 5th day chicks were sacrificed, and

yes were removed and processed for immunocytochemistry (as

escribed below).

Fixation and sectioning. Chicks were sacrificed by chloroform

nhalation. After enucleation, eyes w ere hem isected equatorially,

nd the gel vi tr eous was r emoved f r om the posterior eyecup.

amples were fixed for 30 min at 20C in 4% paraformaldehyde

lus 3% sucrose in 0.1 M phosphate buffer, pH 7.4. Fixed samples

were w ashed t hree t im es in PBS (phosphat e-buffered saline; 0.05 M

phosphat e buffer, 195 mM N aCl, pH 7.4), cryoprotected in PBS plus

30% sucrose, soaked in em bedding medium (O.C.T. compound,

Tissue-Tek) for 10 min, and freeze-mounted onto aluminum sec-

tioning blocks. Transverse sections nominally 14 m thick wer e

cut consistently from the posterior pole of the eye, near the dorsal

portion of the pecten, and thaw-mounted on Super-Fr ost glass

slides (Fisher Scientific). Sections from control an d treated eyes

from the same individual were placed consecutively on each slideto ensure equal exposures to reagents. Sections were air-dried and

stored at 20C until use.

Immunocytochemistry. Sections were w ashed 3 t imes in PBS

covered with primary antibody solution, and incubated for about

24 h at 20C in a humidified chamber . The sl ides wer e washed

three times in PBS, covered with secondary antibody solution (150

l of 1:1500 C y3-conjugated goat anti-rabbit IgG or mouse IgG

Amer sham, and/or 1:400 FITC- conjugated goat anti-r abbit or

mouse IgG, Sigma), and incubated f or about 1 h at 20C in a

humidified chamber. Sections immunolabeled for BrdU or prolif-

erating nuclear antigen (PCNA) were washed in 4 M HCl for 10

m in, followed by th ree more w ashes in PBS (50 mM phosphat e, 145

m M N a C l , p H 7 .4 ) pr io r t o a dd it i on o f t h e p ri m a ry a n t ib od y

solution. Sections doubly labeled for Br dU or PCNA and other

neuron-specific antigens were first labeled with primary antibodies

to the neur on- specific antigen. This was f ollowed by washes in

PBS, labeling with a fluorophore-conjugated secondary antibody

washes in PBS, 10 min in 4 M HCl, washes in PBS, and labeling

with a second primary and secondary fluorophore-conjugated anti-

b od y. Fi n al ly , s am p l es w e re w a sh e d t h r ee t i m e s i n P BS a n d

m oun ted on coverslips in 4:1 (v/v) glycerol to wat er for observation

under an epifluorescence (Nikon Eclipse) or a laser-scanning con-

focal (Bio-Rad H600) microscope by using FITC or rhodam ine fi lter

combinations. Because antigens were not available for preabsorp-

tion controls, we evaluated specificity m ainly by comparison w ith

the results of previous studies using these antibodies and, where

possible, by known hom ologies between th e im mu nizing proteins

and the chick counterparts.

Working dilutions and sources of antibodies used in this study

included mouse anti-BrdU at 1:80 (G3B4; Developmental Studies

Hybridoma Bank, DSHB), rat anti-BrdU at 1:80 (Cappell), mouse

anti-Pax6 at 1:50 (DSHB), rabbit anti-Chx-10 at 1:4000 (Dr. T.

Jessell, C olombia University), rabbit anti-phosphohistone H3 at

1:200 (Upst ate Biotechn ology), m ouse an ti-PCN A at 1:1000 (Dak o),

mouse anti-Hu at 1:200 (Monoclonal Antibody Facility, University

of Oregon), mouse anti-calbindin at 1:1000 (Sigma), rabbit anti-

calretinin at 1:1000 (Dr. J. H. Rogers, University of Cambridge), and

rabbit anti-visinin at 1:1000 (Dr. R.S. Polans, Dow NeurologicalInstitute, Portland, OR).

Labeling of fragmented DNA. Sections of the retinal margin

were obtained as described above from chicks at 1 and 2 days after

NMDA treatment. Slides were washed once in PBS, followed by

one wash in PBS plus 0.3% Tr iton X-100 and 2 mor e washes in

normal PBS. Sections were then covered with 150 l of incubation

medium (0.5 nmol Cy3-conjugated dCTP, 20 units of 3-terminal

deoxynucleotidyl transferase (Amersham), 100 m M sodium caco-

dylate, 2 mM CoCl 2, and 0.25 mM -m ercaptoethanol, in sterile

saline at pH 7.2) and incubated for 1 h in a h um idified chamber at

37C. Sections were th en w ashed th ree tim es in PBS and coverslips

on mounted 4:1 (v/v) glycerol to water for observation by epifluo-

rescence with a rhodamine filter combination.

9 8 Fischer and Reh

Copyright 2000 by Academic Press. All rights of reproduction in any form reserved.


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RESULTS

BrdU Labeling

We probed for mitotically active cells in the chick retina

y u s in g t h e t h y m i di n e a n al og Br dU i n je ct e d i n t o t h e

itreous chamber of the eye. A single dose of BrdU labeled

he occasional cell, one or t wo cells per section in central

etina, in the outer plexiform layer (OPL), inner plexiform

ayer (IPL), ganglion cell layer (GCL), or optic fiber layer

(OFL). Most fields of view of central retina did not contain

any BrdU-labeled nuclei (Fig. 1a). When BrdU was applied

chronically, twice daily for 5 consecutive days, labeled cells

were detected in the OPL and many cells were in the IPL or

OFL (Fig. 1c), consistent with the known position of resi-

dent m icroglia in th e avian retina (N avascue s et al., 1994).

Several BrdU-positive cells (three to seven cells per sec-

tion)were always detected at the retinal margin when BrdU

was applied as a single dose (Fig. 1b). When applied chroni-

IG. 1. BrdU labeling in th e retina of hatched chicks. C entral retina (a) and peripheral retina (b) from a P7 ch ick t hat received a single

ntraocu lar dose of BrdU 4 h prior to enu cleation. Cen tral retin a (c) and peripheral retina (d) from a P7 chick th at received int raocular BrdU

wice daily from P1 through P5. (e) Peripheral retina from a P14 chick that received intraocular BrdU twice daily from P1 through P5. (f)

eripheral retina from a P28 chick that received BrdU once daily from P14 through P19. Arrows in panels b, d, e, and f indicate the retinal

m argin. Abbreviations u sed: GCL, ganglion cell layer; IPL, inn er plexiform layer; IN L, inner nu clear layer; OPL, outer plexiform layer; ON L

uter nuclear layer. Calibration (50 m) in panel e applies to panels a, b, and e; calibration in panel f applies only to that panel; and

alibration in panel d applies to panels c and d.

19 9N eurogenesis in Postnatal Ch icken Retina



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ally, many more cells at the retinal margin were labeled

with BrdU in comparison w ith the num ber of cells labeled

y a s in gl e a pp li ca t io n . W e fo u n d b et w e en 2 0 a n d 4 0

rdU-labeled cells per section n ear th e peripheral m argin of

he retina (Fig. 1d). M ost of t hese cells were at the retinal

m argin, but a few w ere as much as 50 m from the margin

nto th e inn er nu clear layer (INL)an d GC L of th e retina (Fig.

d). BrdU-labeled nuc lei tha t we re c lose to the retina lmargin were oblong or columnar in shape, while labeled

uc lei tha t we re loca te d towa rd the c entra l re tina we re

phere-shaped. In addition, BrdU-positive cells were de-

ected in th e ciliary epithelium, from the retinal m argin t o

bout 200 m toward th e lens (results n ot shown ).

In c hicks tha t s urvive d for 9 days a fte r c hronic BrdU

pplication, labeled cells were displaced centrally from th e

et i n al m a r gi n b y a s m u c h a s 1 0 0 m (Fig. 1e). BrdU-

ositive nuclei in the GCL were located m ore centrally, or

urther from the retinal m argin, t han BrdU-positive nu clei

n the INL (Fig. 1e). Furthermore, these BrdU-labeled nuclei

were spherical in shape, similar to nuclei of fully differen-

ated postmitotic retinal neurons. However, BrdU-labeled

uc le i we re ne ve r s e e n in the oute r nuc le a r la ye r (ONL )

where the nuclei of photoreceptor cells are found. Three

weeks after BrdU t reatm ent , labeled nuclei were found even

urther tow ard the central retina t han t hose observed after 9

ays post BrdU application (result not shown). The abun-

ance of BrdU-labeled nuclei at th e retin al m argin appeared

pproximately equal in retinas that were harvested 1 or 3

weeks after BrdU treatment, suggesting that cells added to

he edge of the retina do not perish after being produced.

To determine wheth er cell proliferation continu es at t he

etinal m argin beyond th e first postnatal w eek, we applied

rdU from P14 to P19 and processed eyes for im m un ocyto-hem istry at P28. In central retina, BrdU-labeled cells were

ot detected (results n ot shown). At the retinal m argin w e

ete c ted ma ny (betwe e n 10 a nd 20 per s ec tion) BrdU-

ositive n uclei; how ever, for equivalen t doses of BrdU fewer

u clei were labeled th an in younger chicks (Fig. 1f). Th ere-

ore , a ddition of ne w c ells to the periphe ral e dge of the

etina continues up to at least 3 weeks after hatching, but

he rate of addition slows as development proceeds.

To confi rm th e presence of dividing cells at th e m argin of

he retina we labeled sections with antibodies to two cell

ycle-related proteins: ant i-phosphohiston e H3 (pHisH3),

which labels cells in the M phase (Mahadevan et al., 1991;hadee et al., 1995; Ajiro et al., 1996), and anti-PCNA. In

he embryonic retina, nu m erous pHisH3-positive cells are

t t he vent ricular surface (Fig. 2a), which is consistent wit h

he known position of M-phase retinal progenitors (Hinds

nd Hinds, 1974). In hatched chicks, we found no cells in

entral retinal regions that were labeled for pHisH3 (Fig.

b); however, we found pHisH3-labeled cells in the ciliary

pithelium and retin al m argin (Fig. 2c). In th e central ret ina

f postnat al chicks, PCN A was absent except for low levels

n the nuclei of photoreceptors (Fig. 2d). However, a small

luster of PCN A-imm unolabeled nuclei was at th e m argin

f the re tina (Fig. 2e ). Immunore a c tivity for PC NA wa s

decreased in cells in the IN L that were located away from

the retinal m argin, wh ile a few cells in t he G CL displaced

from the margin were immunoreactive for PCNA (Fig. 2e).

PCN A-imm unoreactive nu clei were detected in the ciliary

e pithe lium, within 200 m of the retinal margin (results

not s hown). Furthe rmore, PC NA-imm unorea ctive nuc lei

were at the retinal margin and in the ciliary epithelium of

adult birds (4.5 m onths of age) in a pattern similar to thatseen in th e eyes of younger ch icks (Fig. 2f).

Expression of Chx-10 and Pax6

T he a na lys is of c e ll c yc le -re la te d prote ins a nd B rdU

inc orpora tion indic a te d tha t c e ll prolife ra tion oc c urs a t

t h e m a r g i n o f t h e c h i c k e n r e t i n a . T o t e s t w h e t h e r t h e

dividing c ells a t th e re tina l m a rgin of ha tc he d c h ic ks a re

re la te d to e mbryonic re tina l proge nitors , we probe d for

the hom e odom a in t ra ns c ription fa c tors Pax6 a nd C h x-10

These proteins are only coexpressed in embryonic retinalprogenit ors (Belecky-Adam s e t a l . , 1997). Imm un ore ac -

tivities for Pax6 and Chx-10 are present at relatively low

levels in progenitors durin g retinal developm ent (Figs. 3a

and 3b), but becom e highly expressed in different types of

r e t i n a l n e u r o n s a s t h e y d i f f e r e n t i a t e . I n m a t u r e c e n t r a l

r et i n a , s t r on g i m m u n o re ac t iv it y fo r P ax 6 w a s i n t h e

n u c l e i o f a m a c ri n e c el l s, w h i l e w e a k i m m u n o r e ac t i v it y

w a s i n t h e n u c le i o f c el ls i n t h e G C L a n d p r es u m ed

horizont al cells (Fig. 3c), consistent wit h previou s reports

(Belecky-Adams e t a l., 1 9 97 ). Im m u n o r e ac t i vi t y fo r

C h x -1 0 w a s c o m p l i m e n t a r y t o t h a t o f P a x6 , l o c al i ze d

primarily in bipolar cell nuclei (Fig. 3d), consistent with

previous reports (Belecky-Adams et al., 1997). Th ere were

n o c e l l s i n c e n t r a l r e t i n a t h a t c o n t a i n e d b o t h P a x 6 a n d

C h x - 1 0 ( F i g . 3 e ) . I n c o n t r a s t , m a n y c e l l s a t t h e r e t i n a l

m argin coexpressed Pax6 and Ch x-10 (Figs. 3f3h). Many

c e l l s c o l a b e l e d f o r P a x 6 i m m u n o r e a c t i v i t y a n d C h x - 1 0

i m m u n o r ea ct i vi t y w e re a dja ce n t t o t h e n e u r al r et i n a,

c ontiguous with t he c il ia ry e pithe lium, a nd forme d wha t

a p p e a r e d t o b e a p s e u d o s t r a t i fi e d c o l u m n a r e p i t h e l i u m

(Fig. 3h), s imila r to the ge rmina tive ne uroe pithe lium in

the e ye s of a mph ibians (Hollyfie ld, 1968; Stra z nic ky a nd

G a ze , 1 97 1). W h il e C h x -1 0 i m m u n o r ea ct i vi t y i n t h e

c e ntra l re tina wa s c onfine d to bipola r c e lls in the dis ta l

INL , a t the re tina l ma rgin C hx-10-pos it ive nuc le i we re

a ls o found in t he G C L a nd proxima l INL , whe re ga nglion

and amacrine cells are located (Fig. 3g). However, within

10 0 m o f t h e r et i n al m a r gi n , C h x -1 0 w a s n o l on ge r

e x p r e s s e d o u t s i d e o f t h e b i p o l a r c e l l l a y e r o f t h e O N L

(r es u lt s n o t s h ow n ). T h e l oc at i on o f c e ll s t h a t c oe x-

pressed Chx-10 and Pax6 is coincident w ith high levels of

P C N A i m m u n o r e a c t i v i t y a n d B r d U - a c c u m u l a t i n g c e l l s

(Figs . 3i3k). T he s e pa tte rns of la be ling re ma ine d c on-

s ta nt from P0 through P21 a nd we re s imila r in a dult birds

tha t we re 4.5 months of a ge (re s ults not s hown).

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Double-Labeling for BrdU and Markers ofPostmitotic Retinal Neurons

We have shown that mitotically active cells at the retinal

m argin give rise to cells that are integrated int o th e retina. To

etermine whether these newly generated cells differentiate

nto retinal neurons, we doubly labeled retinal sections for

rdU and various neuron-specific markers. One such marker

s th e RN A-binding protein Hu, w hich is expressed soon after

he onset of differentiation in developing neurons (Marusich

t al., 1994). In postnatal chick retina, Hu is expressed by

most, if not all, amacrine and ganglion cells (Fig. 4b). At the

etinal m argin, m any am acrine cells w ere colabeled for BrdU

and Hu immunoreactivity (Figs. 4a4c). We also probed for

BrdU labeling and calretinin imm unoreactivity. In m ature

chick retina calretinin is localized to many amacrine and

ganglion cells, a few bipolar cells, and all h orizont al cells (Ellis

et al., 1991; Fischer et al., 1999). At the retinal margin we

detected some amacrine cells that were colabeled for BrdU

and calret inin im m un oreactivity (Figs. 4d and 4e). Labeling for

BrdU and calbindin immunoreactivity also revealed colocal-

ization. Calbindin is expressed by cone photoreceptors, a

subset of bipolar cells, a subset of amacrine cells, and a few

cells in t he G CL (Ellis et al., 1991). At th e peripheral m argin of

the retina, BrdU was localized to the nuclei of calbindin-

IG. 2. Labeling for PCN A imm unoreactivity and phosphohistine H3 im m unoreactivity in the retinas of hatched chicks. Vertical sections

f retina from (a) E9, (be) P7, and (f) P135 chick. Central retina (b, d) or peripheral retina (c, e, f) were labeled with antibodies to (ac)

hosphoh istone H 3 (pHisH3) or (d, e, f) PCN A. A single m itotic fi gure is labeled for pHisH3 im m un oreactivity in pan el c. Arrows in panels

e, and f indicate the retinal margin. Abbreviations used: GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer;

PL, outer plexiform layer; ONL, outer nuclear layer. Calibration (50 m) in panel c applies to panels a th rough c, calibration in panel e

pplies to panels d and e, and calibration panel f applies only t o panel f.




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mmunoreactive bipolar cells (Figs. 4g4i); however, we did

ot detect BrdU in calbindin-positive photoreceptors. Th is is

onsistent with the finding that BrdU-positive nuclei were

ever seen in the ONL. Nevertheless, we found that some

ells were colabeled for BrdU and the photoreceptor marker

isinin (Figs. 4j4l) (Yamagata et al., 1990). However, th ese

ells did not have the morphology of mature photoreceptors.

BrdU Labeling of Cells at the Retinal Margin of

Form-Deprived Eyes

Fo rm d ep ri va t io n i s k n o w n t o e n la rge t h e e ye a n d,

thereby, cause myopia (reviewed by Wallman, 1993). Exces-

sive eye growth caused by form deprivation is driven by

signals derived from the retina that enhance the addition of

IG. 3. Chick retina immunolabeled for Chx-10, Pax6, and/or PCNA. (a and b) E9 chick retina immunolabeled for (a) Pax6 or (b) Chx-10.

ch) Sections of (ce) central and (fk) peripheral retina that have been doubly labeled for (c, f) Pax6 and (d, g) Chx-10. (ik) Sections of

eripheral retina t hat have been doubly labeled for (i) PCNA and (j) Chx-10. Panels e, h, and k are overlaid composites of th e t wo panels

mm ediately to their left . Yellow indicates doubly labeled nuclei. T he ar rows in panels f thr ough k indicate the r et inal m ar gin

bbreviations used: G CL, ganglion cell layer; IPL, inn er plexiform layer; IN L, inner n uclear. Calibration (50 m ) in panel e applies to panelsth rough e, and calibration in panel k applies to panels f through k.

0 2 Fischer and Reh



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roteoglycans to the posterior pole of the sclera (Rada et al.,

994). In control eyes, that received four consecutive daily

njections of BrdU, on average, between 5 and 10 labeled

ells per section were at the retinal margin (Figs. 5a and 5c).

here were more BrdU-labeled cells in the temporal retinal

ma rgin tha n in the na s al retina l ma rgin (Fig. 5c). Form

eprivation approxim ately doubled th e num ber of BrdU-

abeled cells at the retinal margin. In treated eyes, between

0 a nd 25 BrdU-la be le d c e lls we re found a t the retina l

m argin (Figs. 5b and 5c). These BrdU-labeled cells were

ocated up to 80 m into th e retina, away from t he m argin.

imilar to controls, there were more BrdU-labeled cells in

the t emporal margin th an in t he nasal margin of the retina

of form-deprived eyes (Fig. 5c). While visual deprivation

increased the nu m ber of BrdU-labeled cells th at were added

to t he retina, there was no change in t he n um ber of labeled

c ells tha t we re in the c il ia ry e pithe lium a dja ce nt to the

retinal margin (Fig. 5c).

T o c orroborate findings of BrdU-la be ling s tudie s, we

probed the retinal margin of form-deprived eyes with anti-

bodie s to PC NA. Afte r 5 da ys of vis ua l de priva tion, the

population of PCNA-immunoreactive cells at the retinal

ma rgin wa s s ubsta ntially inc rea se d c ompa red to tha t of

controls (Figs. 5e and 5d).

IG. 4. Sections of chick retinal m argin th at were double-labeled for BrdU and other m arkers comm on to postm itotic retin al neurons. Eyes

ere exposed t o BrdU t wice daily from P1 th rough P5 an d enu cleated on P14. Sections were dou bly labeled for (a, d, g, j) BrdU an d (b) Hu ,

e) calretinin, (h) calbindin, or (k) visinin. Images in panels a through c were obtained using a laser confocal microscope. In all panels, the

etinal margin is to the left and the photoreceptor layer is to the top. Arrows indicate doubly labeled cells. Panels c, f, i, and l are overlaid

omposit es of the t wo panels im m ediately to t heir left. Abbreviations used: GCL, ganglion cell layer; IPL, inner plexiform layer; IN L, inn er

uclear. Calibration (50 m) in panel c applies to panels a through c, and calibration in panel l applies to panels d th rough l.




8/14

Effects of Acute Damage on Proliferating Cells athe Retinal Margin

Excitotoxic c ell dea th c aus es a large inc re as e in the

rolife ra tion of proge nitors a t the retina l ma rgin of th e

adpole retina (Reh, 1987). T o test wheth er progenitors at

he margin of the postnatal chick retina respond to acute

amage with increased proliferation, we injected a single

oxic dos e of N M DA into t he vitre ous c ham ber of ne wly

a tc he d c hic ks . NM DA de s troys ma ny bipola r a nd a ma -

rine cells in the central retina of the chick, as indicated by

DN A-fragme nta tion labeling, a s ym ptom of a poptos is

Fischer et al., 1998). In saline-treated retin as, a few n uclei

per section) labeled for DNA fragmentation were detected

n the INL within 100 m of the retinal m argin (Fig. 6a),

suggesting that some newly generated cells may not sur-

vive. Many nuclei in the INL were labeled for fragm ented

D N A a t t h e p er ip h er al m a r gi n o f r e t in a s t r ea t ed w i t h

NMDA (Fig. 6b), indicating that there was damage and cell

death in this region. Surprisingly, there was no increase in

the number of BrdU-labeled cells at the margin of toxin-

treated retinas (Fig. 6d) compared to the number of labeled

cells seen in controls (Fig. 6c). There was, h owever, an

inc re as e in the num ber of BrdU-labeled c ells s ca tte re d

across the IPL (results not shown). These cells were likely

to be activated microglia, consistent with the known loca-

tion of phagocytes in N MDA-damaged retinas (Fischer et

al., 1998). These findings suggest that the rate of prolifera-

tion of progenitors in the marginal zone of the chick retina

IG. 5. Effects of visual deprivation on the addition of BrdU-labeled cells to the peripheral margin of the chick retina. Vertical sections

f chick retina that were obtained from eyes injected once daily for 4 consecutive days with BrdU from P1 through P4 and (a) left open or

) form-deprived from P0 t hrough P5. Calibration 50 m . The histogram in panel c illustrates the nu m ber of BrdU-labeled cells in t he

iliary epithelium adjacent to the retinal margin and in the INL at the margin of the retina of open or form-deprived eyes. Statistical

gnifi cance (* P 0.05; ** P 0.0 05) was assessed by using a paired tw o-tailed Stu dent s t test . Error bars represent t he stan dard deviation

f the sample m ean (N 6). (d and e) PCN A im mu nolabeling in vertical sections of the retinal m argin obtained from eyes that were (d)

eft open or (e) form-deprived from P0 through P5. Calibration 50 m .

0 4 Fischer and Reh



9/14

s u ne ffe cte d by dama ge, unlike the proge nitors in the

marginal zone of the tadpole retina (Reh, 1987).

Effects of Grow th Factors on Proliferating Cells athe Retinal Margin

In the goldfish retina, insulin-related growth factors in-

reased the proliferation of progenitors at the retinal m ar-

in, while other growth factors had no effect (Boucher and

Hitchcock, 1998). Accordingly, we tested whether retinal

rogenitors at the m argin of the avian retina proliferate in

esponse to growt h factors. Proliferating cells, labeled wit h

rdU, were in tw o distinct populat ions of cells; th ose at th e

etinal margin (Figs. 7a7f) and those in the ciliary epithe-um (Fig. 7g). We foun d t hat 100 n g per dose of EGF, IGF-I,

nd insulin induced proliferation of retinal progenitors at

he margin, while 10-ng doses or bFGF at either dose did

ot. Four consecutive daily injections of 10 ng of EGF had

o e ffe ct upon the num ber of BrdU-labeled c ells in the

etina l m a rgin or in the c il ia ry e pithe lium (res ults not

hown). Two consecutive daily injections of 100 ng EGF

e arly t riple d the num ber of BrdU-labeled c ells a t the

etinal margin (Figs. 7b and 7f). Similar to EGF, 10 ng of

FGF had no effect upon the numbers of cells at the retinal

m argin t hat were labeled with BrdU (results n ot sh own). An

ncreased dosage of bFGF (100 ng) had no effect upon the

numbers of BrdU-labeled cells at the retinal margin and

ciliary epithelium (Figs. 7c and 7f). However, this dose of

bFGF caused a large increase in the number of BrdU-labeledcells in the apical tips of ciliary protrusions (results not

s hown), indic ating t ha t this dos e of growth fa ctor wa s

effective, but did not influ ence th e proliferation of progeni-

tors at the retinal margin. Four consecutive daily applica-

t i on s o f 1 0 n g I G F-I h a d n o e ffe ct u p on t h e n u m b e r o f

BrdU-labeled cells at the retinal margin or in the adjacent

ciliary epithelium (results not shown). However, two con-

secutive daily injections of 100 ng IGF-I more than tripled

the number of BrdU-labeled cells at the retinal margin (Figs.

7d and 7f). Consistent with this result, 500 ng of insulin

greatly increased th e num ber of BrdU-labeled cells in the

ciliary epithelium and retinal margin (Figs. 7e and 7f).To corroborate these findings we probed for PCNA im-

m unoreactivity at the retinal m argin of eyes injected with

growth factors. Consistent with findings of BrdU labeling,

w e fo u n d t h a t EG F, IG F-I, a n d i n su l in , b u t n o t b FG F,

increased t he population of cells at the retinal m argin t hat

are immunoreactive for PCNA (Fig. 8). This increase in

PCNA immunolabeling was detected at P7, 4 days after the

application of growth factors. Assum ing that growt h factors

are cleared from th e eye within 24 h after application, these

findings suggest that growth factors have a residual, long-

lasting effect on proliferation or that PCNA is expressed

long after cells have completed their cycle.

IG. 6. NMDA-induced damage at the retinal margin does not effect the production of new cells. Vertical sections of retinal margin were

abeled for (a, b) fragment ed DN A or (c, d) BrdU im m un oreactivity. Eyes were in jected at P0 with (a, c) saline or (b, d) 2 m o l N M D A a n d

arvested at P7 following injection of 0.5 g BrdU at P2 and P3. Arrows indicate t he retinal m argin. Calibration 50 m .




10/14

DISCUSSION

Here we provide evidence that a proliferating marginal

one , c onta ining c e lls tha t re se mble mu ltipote nt e mbry-

nic progenitors, is present in the retina of postembryonic

warm-blooded vertebrates. Our evidence indicates that: (i)

roliferation of cells cont inu es into adult hood at th e retinal

margin of the chicken eye; (ii) cells at the retinal margin

c oe xpres s Pa x6, C hx-10, a nd PC NA a nd a re s im ila r to

m ultipotent retinal progenitors; and (iii) these retinal pro-

genitors produce new neurons that are integrated into the

edge of the retina. The proliferating marginal zone of the

c hic k re tina is s imila r to tha t of fis h a nd a mphibia ns in

which a zone of stem cells produces new retinal neurons

IG. 7. BrdU labeling at the retinal margin after treatment with various growth factors. Vertical sections of the chick retinal margin were

btained from P7 eyes that were injected on P2 and P3 w ith 0.5 g BrdU plu s (a) saline, (b) 100 n g EGF, (c) 100 n g bFGF, (d) 100 n g IGF-I

r (e) 500 n g insulin. Arrows in dicate the retinal m argin. C alibration 50 m. The histograms in panels f and g illustrate the effects of

aline, EGF, bFGF, IGF-I, and in sulin on t he n um ber of BrdU-labeled cells in (f) th e tem poral and n asal retinal m argin an d (g) th e tem poral

nd nasal ciliary epithelium (300 m adjacent to t he ret inal m argin). Significance of difference (* P 0.05; ** P 0.001; *** P 0 . 0 0 0 5 )

etween saline- and growth factor-treated eyes was determined by using a two-tailed Students t test. Error bars represent the standard

eviation of the sample mean (N 6 ).

0 6 Fischer and Reh



11/14

hroughout life (Hitchcock and Raymond, 1993; Raymond

nd Hitc hcock, 1997; Hollyfie ld, 1968; Stra znic ky a nd

Gaze, 1971; Fernald, 1990). While progenitors in the am-

hibian retina increase t heir proliferation and regenerateeuron s in response to dam age (Reh, 1987), th e destruct ion

f cells at the margin of the chick retina does not induce

rogenitors to increase their rate of proliferation.

The fi nding that the chicken retina contains a proliferat-

ng marginal zone came as a surprise to us, not because it

ontradicts prior reports, but rather because these findings

ave gone un not iced and it h as been generally assum ed that

h e r et i n a o f h a t c h ed c h ic k s d oe s n ot h av e a z on e o f

roliferating cells at the peripheral margin. Morris e t a l .

1976) provided a brief report that [H 3]thymidine accumu-

ates in some cells at t he m argin of the chick retina during

he fi rst 2 postnatal weeks. Other reports on retinal neuro-enesis have focused on the birth dates of different retinal

ell types (Fujita and Horii, 1963; Kahn, 1973, 1974; Spence

nd Robson, 1989; Prada e t a l. , 1991), with only s ome

tten tion paid to t he en d of proliferation (Prada et al., 1991).

However, the retinal margin has a regenerative capacity in

ate-stage embryos (Willbold and Layer, 1991), which is

onsistent with our observations.

In central retina, very few BrdU-labeled cells were de-

ected even when BrdU was applied repeatedly, suggesting

hat these cells are not abundant or that very few of them

re dividing. The identity of BrdU-positive cells in the OPL,

PL, a nd OFL rema ins unc e rta in. T he s hape, s iz e, a nd

location of these nuclei are characteristic of resident, non-

activat ed m icroglia in avian r etin a (N avascue s et al., 1994).

Additionally, some of the BrdU-labeled cells in the GCL

and OFL could be oligodendroglia. In the chick, m yelina-tion of ganglion cell axons in t he O FL occurs after h atchin g

(Rager, 1976; Arees, 1978) and may be coincident with glial

proliferation, as su ggested by BrdU labeling in th e G CL and

OFL (current study). The generation of these put ative glia is

completed before P14 since BrdU, applied daily from P14

though P19, did not label these cells.

In eyes that were harvested several days after BrdU was

applied, labeled cells were found away from the peripheral

edge of the retina, suggesting that subsequent addition of

cells had occurred to displace these cells from the margin.

Colabeling for BrdU and neuronal markers was found only

in re tina l c e lls tha t we re loc a te d a wa y from the ma rgin.Furthermore, large numbers of cells were detected at the

retinal m argin only w hen BrdU was applied repeatedly.

These fin dings suggest th at progenitors at t he retinal m ar-

gin are dividing and differentiating very slowly and that

there is concentric addition of new neurons to the periph-

eral edge of the retina.

Th e integration of neuron s into th e retina and acquisition

of ne ural phe notypes occ urre d m uc h more s lowly tha n

during e mbryonic de ve lopme nt. We found tha t a bout 80

m of radial length of retina was added in 2 weeks (P0 to

P14) of postnatal development , while m ore than 100 tim es

t h i s l en gt h w a s f or m e d i n a bo u t 1 2 d ay s o f e m b ry on i c

IG. 8. PCN A labeling at th e retinal margin after treatm ent w ith various growth factors. Vertical sections of th e chick retinal m argin were

btained from P7 eyes that were injected on P2 and P3 w ith 0.5 g BrdU plu s (a) saline, (b) 100 n g EGF, (c) 100 n g bFGF, (d) 100 n g IGF-I

r (e) 500 ng insulin. Arrows indicate t he retinal m argin. C alibration 50 m .




12/14

evelopment (Kahn, 1973, 1974; Prada et al., 1991). Al-

hough the c ha ra cte r of e mbryonic a nd pos tna tal retina l

roge nitors is s imila r, the ir mitotic a c tivity a nd ra te of

euron production differ greatly. It seems likely that post-

a ta l re tina l proge nitors do not rec eive th e s t imula tory

rowth fa ctors th a t e nha nce the ir rate s of division a nd

roduction of new neu rons. This hypoth esis is supported by

ur findings tha t e xoge nous ins ulin, IGF-I, a nd EGF in-re a s e the prolife ra tion a nd a ddition of ne w c e lls to the

etinal m argin. Th ese findings indicate that progenitors at

he re tina l m a rgin a re c om pete nt to res pond to growth

a c tors , but tha t the c onc e ntra tion of the fa c tors ma y be

ery low in t he postnat al eye. Alternatively, th e preexisting

etinal neurons at the margin may secrete inhibitory factors

hat suppress the activity of these progenitors.

While the re a re ma ny ne wly ge ne ra te d ne urons a t the

etinal m argin, few of th ese cells were colabeled for visinin

mmunoreactivity and BrdU. These colabeled cells did not

ppear to be differentiated photoreceptors, but may have

een in an in term ediate state of differentiation . In addition,

rdU-labeled nuclei were never found in the ONL, where

he nuc le i of photore c e ptors a re loc a te d. T he s e findings

uggest that new cone photoreceptors may not be produced

t the retinal margin. Since this cell type is formed rela-

vely early during em bryonic development (Prada et al.,

991; Spence and Robson, 1989), it is possible th at t he cu es

equire d for the ge ne ra tion of c one photorec eptors a re

bsent in postnatal birds. While BrdU was found in ama-

rine and bipolar cells, BrdU w as not foun d in h orizontal or

anglion cells. BrdU-labeled nuclei w ere found in th e GC L,

ut these cells did not express Hu . It is possible that these

ells m ight be dis plac ed a ma c rine c ells (tha t ma y not

xpress Hu ) or a t ype of ganglion cell th at does n ot expressHu. Taken together, these findings suggest that amacrine

nd bipolar c ells , but not photorec eptor, horiz onta l , or

anglion cells, are produced by progenitors at the retinal

m argin of postnatal chicks.

The effects of different growth factors on the proliferation

f progenitors at t he retinal m argin are consistent with the

ndings of studies on embryonic retinal progenitors and

tem cells in oth er anim als. EGF receptor ligands have been

hown to stimulate proliferation of retinal progenitors in

odents (Anchan et al., 1991; Lillien and Cepko, 1992) and

romot e differentiation of am acrine an d M uller cells (An-

han and Reh, 1995; Lillien, 1996). It is possible th at, in th eostnatal ch ick, exogenous EGF not only indu ced prolifera-

on of retinal progenitors but also increased numbers of

ells that differentiat ed as am acrine and Muller cells. Con -

istent with this h ypothesis, we observed th at m ost BrdU-

abeled cells in EGF-treated retinas were within the INL,

wh ere differentiated am acrine and Muller cells are k nown

o reside. However, in the absence of EGF, BrdU-labeled

uclei accum ulate primarily in t he INL and, th erefore, we

annot say th at cell fate w as influenced.

Other growth factors used in this study have been shown

lsewhere to be mitogenic for retinal progenitors. For ex-

mple, insulin and IGFs increase the proliferation of stem

cells at the peripheral margin of the goldfish retina (Boucher

and Hitchcock, 1998). However, it is puzzling that bFGF

was not m itogenic for t hese cells, since it is a m itogen for

progenitors in th e developing rat ret ina (Lillien and C epko,

1992; Anchan and Reh, 1995). Many studies have demon-

strated the importance of FGFs to normal eye development

in ch ick em bryos (Park an d Hollenberg, 1989; Pittack et al.,

1991; Pittacket al.,

1997). Thus, there appear to be simi-larities and differences in types of secreted factors that

stimulate proliferation of retinal progenitors in fish, birds,

a nd m a m m a ls . H o w ev er , i t i s w o r th n ot i ng t h at m o st

studies of mitogens in fish and m amm als have used in vitro

a s s a ys , while in the pre s e nt s tudy we ha ve a na lyz e d the

effects of th ese factors in vivo.

The addition of new neurons to the margin of the retina

m ay serve to com pensate, at least in part, for retinal stret ch

that is imposed during postnatal ocular growth. When rates

of ocula r growth we re inc re as ed by vis ua l deprivation,

greater numbers of BrdU-labeled cells were added to the

retinal margin. Since excessive ocular growth induced by

visual deprivation results from the addition of proteogly-

cans to the posterior pole of th e sclera (Rada et al., 1994),

th e retina becom es stretched as th e eye grows too m uch and

becomes myopic (Teakle et al., 1993). During normal post-

natal growth and excessive growth induced by visual depri-

va tion re tina l s tre tc h ma y induc e the proge nitors a t the

margin to increase their rate of division. We propose that

mechanical stretch at the retinal margin increased the rates

of division of proge nitors. In c om paris on, m e cha nica l

stretch increases the proliferation of cardiac fibroblasts

(Booz and Baker, 1995) and cells of the nucleus pulposus

(Matsumoto et al., 1999).

In fish and amphibians, retinal stretch and the addition ofnew neurons compensate, in part, for decreased neuronal

density per unit area of retina that results from overall eye

growth during postembryonic development (Fernald, 1990;

Hitchcock and Raymond, 1993; Raymond and Hitchcock,

1997). Althou gh stretching of central retina occurs during

postnatal ocular growth (Teakle et al., 1993), the addition of

new neurons at the retinal margin of the chick may serve to

c om p e n sa t e i n p ar t fo r t h i s p h en o m e n on . T h i s a n n u la r

a ddition of ne w ne urons to the pe riphe ra l ma rgin of the

c hick retina is s im ila r to the ongoing ne uroge ne ra tive

process that occurs in the eyes of lower vertebrates.

ACKNOWLEDGMENTS

We thank Paulette Brunner at the Kocke Imaging Facility and

Blair Dierks for their expert technical assistance. We also thank

Drs. W. K. Stell, E. M. Levine, and A. Davis for their com m ents th at

helped to contribute t o the final form of this paper. The BrdU and

Pax6 antibodies developed by Drs. S. J. Kaufman and A. Kawakam i,

respectively, were obtained from the Developmental Studies Hy-

bridoma Bank developed un der auspices of the N ICHD and main-

tained by The University of Iowa, Department of Biological Sci-

ences, Iowa City, Iowa 52242. We gr atefully acknowledge the

contribution of Myron and Arletta Rasmussen to this study. This

0 8 Fischer and Reh



13/14

or k was suppor ted by fellowships fr om the Alberta Her itage

oundation for Medical Research, the Medical Research Council of

anada, and The Fight For Sight Foundation Research Division of

revent Blindness Am erica to A.J.F. and by an N SF grant (IBN

64843), and the Foundation Fighting Blindness to T.A.R.

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Andy J. Fischer and Thomas A. Reh- Identification of a Proliferating Marginal Zone of Retinal Progenitors in Postnatal Chickens