Developmental Biology 220, 197210 (2000) doi:10.1006/dbio.2000.9640, available online at http://www.idealibrary.

com on

Identication of a Proliferating Marginal Zone of Retinal Progenitors in Postnatal Chickens

Andy J. Fischer and Thomas A. Reh 1
Department of Biological Structure, University of Washington, Seattle, Washington 98195

In warm-blooded vertebrates it is generally accepted that after early stages of development new neurons are not added to the retina. Contrary to this belief, we show here that hatched chickens have a zone of proliferating cells at the peripheral margin of the retina, similar to that of sh and amphibians. We found that cells at the peripheral edge of the retina incorporated the thymidine analog BrdU and expressed the cell cycle regulator proliferating cell nuclear antigen (PCNA). Furthermore, cells in the ciliary epithelium and retinal margin coexpressed the homeodomain transcription factors Pax6 and Chx-10, similar to 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 expressed proteins found only in differentiated neurons. Increased rates of ocular growth, induced by visual deprivation, resulted in increased numbers of BrdU-labeled cells at the retinal margin. Unlike the progenitors in the retinal marginal zone of sh and amphibians, the progenitors of the chick retina do not increase their rate of proliferation in response to acute damage. Furthermore, insulin, insulin-like growth factor-I, and epidermal growth factor increased proliferation of progenitors at the retinal margin, while basic broblast growth factor had no effect. These results indicate that the avian retina has a marginal growth zone containing proliferating cells that share similarities with multipotent embryonic retinal progenitors 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.

INTRODUCTION
Retinal histogenesis in warm-blooded vertebrates (birds and mammals) is thought to occur only during early stages of development. For example, all cell types in the chick retina are believed to be formed more than 1 week before hatching (Prada et al., 1991). Differentiation of cells begins around Embryonic Day 2 (E2) in the central retina and progresses to peripheral regions. The rst cells formed during retinal neurogenesis are the ganglion cells (Kahn, 1973), followed closely by photoreceptors, amacrine and horizontal cells (Fujita and Horri, 1963; Morris, 1973; Spence and Robson, 1989; Prada et al., 1991). The last cell types formed are the bipolar cells and Muller glia, with the nal cell divisions in the retina reported to occur around E12 (Kahn, 1974; Prada et al., 1991). The chick retina is fully functional and believed to be postmitotic at the time of hatching, about E21. However, Morris et al. (1976) provided a brief account that [H 3]thymidine accumulates in
1 To whom correspondence should be addressed. Fax: (206) 5431524. E-mail: tomreh@u.washington.edu.

some cells at the extreme periphery of the postnatal chick retina. Using modern techniques and reagents, here we tested whether neurogenesis continues at the retinal margin of hatched chicks. During postnatal development, the eye continues to grow in parallel with overall body size, and most vertebrates, including humans, are born hyperopic with their eyes too small relative to the refractive power of the lens and cornea (Curtin, 1985). Postnatally, when the eyes rst receive clear images, rates of ocular growth are increased, regulated by retinal processing of visual information, so that eye size is precisely matched to refractive power (Wallman, 1993). Ocular shape and size are determined by the growth of the sclera, the outer connective tissue sheath of the eye. In amphibians and teleost sh, growth of the eye is coordinated with the addition of new neurons at the peripheral margin of the retina and, in the case of teleosts, new rod photoreceptors in central regions (Johns and Fernald, 1981; Johns, 1982). In some cold-blooded vertebrates, new neurons at the retinal margin are generated by a population of stem cells that persist throughout life (reviewed by Hitchcock and Raymond, 1993; Raymond and Hitchcock, 1997;

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Reh and Levine, 1998; Perron et al., 1998). In birds and mammals, however, postnatal ocular growth is not believed to be coincident with the addition of new neurons at either the margin or the central regions of the retina. Instead, a passive stretching of retina is thought to occur to compensate for eye growth (Teakle et al., 1993). Here we provide evidence that in postnatal chicks there is a proliferating marginal zone containing cells that share similarities with embryonic retinal progenitors and the retinal stem cells of sh and amphibians. This zone produces new neurons during adolescent ocular growth and is present in adult animals. We also show that the proliferation of progenitors at the retinal margin can be increased by enhancing rates of ocular growth or by applying a variety of growth factors.

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METHODS AND MATERIALS

Animals. The use of animals in these experiments was in accordance with the guidelines established by the National Institutes of Health and the University of Washington. Newly hatched leghorn chickens (Gallus gallus domesticus) were obtained from H&N Highline International (Seattle, WA) and kept on a cycle of 16 h light, 8 h dark (lights on at 6:00 am). Chicks were housed in clear Nalgene cages at about 25C and received water and Purina chick starter ad libitum. Injections. Intraocular injections of BrdU were used to label dividing cells in the retina. Chicks were anesthetized with halothane prior to injection. Injections were made into the vitreous chamber using a 25- l Hamilton syringe with a 26-gauge needle. Penetration of the needle was made consistently into the dorsal half of the eye. The left eye (control) was injected with 20 l of sterile saline and the right eye (treated) was injected with BrdU (between 0.5 and 10 g) dissolved in sterile saline. To test whether growth factors inuence the proliferation of cells at the retinal margin we injected eyes at P2 and P3 with BrdU (0.5 g) and different growth factors, and harvested eyes at P7 to process them for BrdU immunoreactivity. Growth factors used in these experiments included puried bovine insulin (500 ng), recombinant human insulin-like growth factor-I (IGF-I, between 1 and 100 ng per injection), recombinant human epidermal growth factor (EGF, 10 or 100 ng per injection), and puried bovine basic broblast growth factor (bFGF, 10 or 100 ng per injection). All growth factors were obtained from R & D Systems and were dissolved in saline plus 0.1 mg/ml BSA and 0.5 g BrdU. In another set of experiments, a toxic dose of N-methyl-D-aspartate (NMDA; 280 g) was injected into the left eye of newly hatched chicks, followed by four consecutive daily injections of 0.5 g of BrdU. All drugs were obtained from Sigma. Visual deprivation experiments. Newly hatched chicks were form-deprived by gluing a translucent light-diffusing goggle over their left eye. In six chicks, injections of BrdU (1.0 g/dose) were made into the vitreous chamber (as described above) of both eyes for 4 consecutive days. On the 5th day chicks were sacriced, and eyes were removed and processed for immunocytochemistry (as described below). Fixation and sectioning. Chicks were sacriced by chloroform inhalation. After enucleation, eyes were hemisected equatorially, and the gel vitreous was removed from the posterior eyecup. Samples were xed for 30 min at 20C in 4% paraformaldehyde plus 3% sucrose in 0.1 M phosphate buffer, pH 7.4. Fixed samples

were washed three times in PBS (phosphate-buffered saline; 0.05 M phosphate buffer, 195 mM NaCl, pH 7.4), cryoprotected in PBS plus 30% sucrose, soaked in embedding medium (O.C.T. compound, Tissue-Tek) for 10 min, and freeze-mounted onto aluminum sectioning blocks. Transverse sections nominally 14 m thick were cut consistently from the posterior pole of the eye, near the dorsal portion of the pecten, and thaw-mounted on Super-Frost glass slides (Fisher Scientic). Sections from control and treated eyes from the same individual were placed consecutively on each slide to ensure equal exposures to reagents. Sections were air-dried and stored at 20C until use. Immunocytochemistry. Sections were washed 3 times in PBS, covered with primary antibody solution, and incubated for about 24 h at 20C in a humidied chamber. The slides were washed three times in PBS, covered with secondary antibody solution (150 l of 1:1500 Cy3-conjugated goat anti-rabbit IgG or mouse IgG, Amersham, and/or 1:400 FITC-conjugated goat anti-rabbit or mouse IgG, Sigma), and incubated for about 1 h at 20C in a humidied chamber. Sections immunolabeled for BrdU or proliferating nuclear antigen (PCNA) were washed in 4 M HCl for 10 min, followed by three more washes in PBS (50 mM phosphate, 145 mM NaCl, pH 7.4) prior to addition of the primary antibody solution. Sections doubly labeled for BrdU or PCNA and other neuron-specic antigens were rst labeled with primary antibodies to the neuron-specic antigen. This was followed by washes in PBS, labeling with a uorophore-conjugated secondary antibody, washes in PBS, 10 min in 4 M HCl, washes in PBS, and labeling with a second primary and secondary uorophore-conjugated antibody. Finally, samples were washed three times in PBS and mounted on coverslips in 4:1 (v/v) glycerol to water for observation under an epiuorescence (Nikon Eclipse) or a laser-scanning confocal (Bio-Rad H600) microscope by using FITC or rhodamine lter combinations. Because antigens were not available for preabsorption controls, we evaluated specicity mainly by comparison with the results of previous studies using these antibodies and, where possible, by known homologies between the immunizing 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, Colombia University), rabbit anti-phosphohistone H3 at 1:200 (Upstate Biotechnology), mouse anti-PCNA at 1:1000 (Dako), mouse anti-Hu at 1:200 (Monoclonal Antibody Facility, University of Oregon), mouse anti-calbindin at 1:1000 (Sigma), rabbit anticalretinin at 1:1000 (Dr. J. H. Rogers, University of Cambridge), and rabbit anti-visinin at 1:1000 (Dr. R.S. Polans, Dow Neurological Institute, 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% Triton X-100 and 2 more 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 mM sodium cacodylate, 2 mM CoCl 2, and 0.25 mM -mercaptoethanol, in sterile saline at pH 7.2) and incubated for 1 h in a humidied chamber at 37C. Sections were then washed three times in PBS and coverslips on mounted 4:1 (v/v) glycerol to water for observation by epiuorescence with a rhodamine lter combination.

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FIG. 1. BrdU labeling in the retina of hatched chicks. Central retina (a) and peripheral retina (b) from a P7 chick that received a single intraocular dose of BrdU 4 h prior to enucleation. Central retina (c) and peripheral retina (d) from a P7 chick that received intraocular BrdU twice daily from P1 through P5. (e) Peripheral retina from a P14 chick that received intraocular BrdU twice daily from P1 through P5. (f) Peripheral 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 margin. Abbreviations used: GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer 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 calibration in panel d applies to panels c and d.

RESULTS
BrdU Labeling
We probed for mitotically active cells in the chick retina by using the thymidine analog BrdU injected into the vitreous chamber of the eye. A single dose of BrdU labeled the occasional cell, one or two cells per section in central retina, in the outer plexiform layer (OPL), inner plexiform layer (IPL), ganglion cell layer (GCL), or optic ber layer

(OFL). Most elds 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 resident microglia in the avian retina (Navascues et al., 1994). Several BrdU-positive cells (three to seven cells per section) were always detected at the retinal margin when BrdU was applied as a single dose (Fig. 1b). When applied chroni-

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cally, many more cells at the retinal margin were labeled with BrdU in comparison with the number of cells labeled by a single application. We found between 20 and 40 BrdU-labeled cells per section near the peripheral margin of the retina (Fig. 1d). Most of these cells were at the retinal margin, but a few were as much as 50 m from the margin into the inner nuclear layer (INL) and GCL of the retina (Fig. 1d). BrdU-labeled nuclei that were close to the retinal margin were oblong or columnar in shape, while labeled nuclei that were located toward the central retina were sphere-shaped. In addition, BrdU-positive cells were detected in the ciliary epithelium, from the retinal margin to about 200 m toward the lens (results not shown). In chicks that survived for 9 days after chronic BrdU application, labeled cells were displaced centrally from the retinal margin by as much as 100 m (Fig. 1e). BrdUpositive nuclei in the GCL were located more centrally, or further from the retinal margin, than BrdU-positive nuclei in the INL (Fig. 1e). Furthermore, these BrdU-labeled nuclei were spherical in shape, similar to nuclei of fully differentiated postmitotic retinal neurons. However, BrdU-labeled nuclei were never seen in the outer nuclear layer (ONL) where the nuclei of photoreceptor cells are found. Three weeks after BrdU treatment, labeled nuclei were found even further toward the central retina than those observed after 9 days post BrdU application (result not shown). The abundance of BrdU-labeled nuclei at the retinal margin appeared approximately equal in retinas that were harvested 1 or 3 weeks after BrdU treatment, suggesting that cells added to the edge of the retina do not perish after being produced. To determine whether cell proliferation continues at the retinal margin beyond the rst postnatal week, we applied BrdU from P14 to P19 and processed eyes for immunocytochemistry at P28. In central retina, BrdU-labeled cells were not detected (results not shown). At the retinal margin we detected many (between 10 and 20 per section) BrdUpositive nuclei; however, for equivalent doses of BrdU fewer nuclei were labeled than in younger chicks (Fig. 1f). Therefore, addition of new cells to the peripheral edge of the retina continues up to at least 3 weeks after hatching, but the rate of addition slows as development proceeds. To conrm the presence of dividing cells at the margin of the retina we labeled sections with antibodies to two cell cycle-related proteins: anti-phosphohistone H3 (pHisH3), which labels cells in the M phase (Mahadevan et al., 1991; Chadee et al., 1995; Ajiro et al., 1996), and anti-PCNA. In the embryonic retina, numerous pHisH3-positive cells are at the ventricular surface (Fig. 2a), which is consistent with the known position of M-phase retinal progenitors (Hinds and Hinds, 1974). In hatched chicks, we found no cells in central retinal regions that were labeled for pHisH3 (Fig. 2b); however, we found pHisH3-labeled cells in the ciliary epithelium and retinal margin (Fig. 2c). In the central retina of postnatal chicks, PCNA was absent except for low levels in the nuclei of photoreceptors (Fig. 2d). However, a small cluster of PCNA-immunolabeled nuclei was at the margin of the retina (Fig. 2e). Immunoreactivity for PCNA was

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decreased in cells in the INL that were located away from the retinal margin, while a few cells in the GCL displaced from the margin were immunoreactive for PCNA (Fig. 2e). PCNA-immunoreactive nuclei were detected in the ciliary epithelium, within 200 m of the retinal margin (results not shown). Furthermore, PCNA-immunoreactive nuclei were at the retinal margin and in the ciliary epithelium of adult birds (4.5 months of age) in a pattern similar to that seen in the eyes of younger chicks (Fig. 2f).

Expression of Chx-10 and Pax6

The analysis of cell cycle-related proteins and BrdU incorporation indicated that cell proliferation occurs at the margin of the chicken retina. To test whether the dividing cells at the retinal margin of hatched chicks are related to embryonic retinal progenitors, we probed for the homeodomain transcription factors Pax6 and Chx-10. These proteins are only coexpressed in embryonic retinal progenitors (Belecky-Adams et al., 1997). Immunoreactivities for Pax6 and Chx-10 are present at relatively low levels in progenitors during retinal development (Figs. 3a and 3b), but become highly expressed in different types of retinal neurons as they differentiate. In mature central retina, strong immunoreactivity for Pax6 was in the nuclei of amacrine cells, while weak immunoreactivity was in the nuclei of cells in the GCL and presumed horizontal cells (Fig. 3c), consistent with previous reports (Belecky-Adams et al., 1997). Immunoreactivity for Chx-10 was complimentary to that of Pax6, localized primarily in bipolar cell nuclei (Fig. 3d), consistent with previous reports (Belecky-Adams et al., 1997). There were no cells in central retina that contained both Pax6 and Chx-10 (Fig. 3e). In contrast, many cells at the retinal margin coexpressed Pax6 and Chx-10 (Figs. 3f3h). Many cells colabeled for Pax6 immunoreactivity and Chx-10 immunoreactivity were adjacent to the neural retina, contiguous with the ciliary epithelium, and formed what appeared to be a pseudostratied columnar epithelium (Fig. 3h), similar to the germinative neuroepithelium in the eyes of amphibians (Hollyeld, 1968; Straznicky and Gaze, 1971). While Chx-10 immunoreactivity in the central retina was conned to bipolar cells in the distal INL, at the retinal margin Chx-10-positive nuclei were also found in the GCL and proximal INL, where ganglion and amacrine cells are located (Fig. 3g). However, within 100 m of the retinal margin, Chx-10 was no longer expressed outside of the bipolar cell layer of the ONL (results not shown). The location of cells that coexpressed Chx-10 and Pax6 is coincident with high levels of PCNA immunoreactivity and BrdU-accumulating cells (Figs. 3i3k). These patterns of labeling remained constant from P0 through P21 and were similar in adult birds that were 4.5 months of age (results not shown).

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FIG. 2. Labeling for PCNA immunoreactivity and phosphohistine H3 immunoreactivity in the retinas of hatched chicks. Vertical sections of retina from (a) E9, (b e) P7, and (f) P135 chick. Central retina (b, d) or peripheral retina (c, e, f) were labeled with antibodies to (a c) phosphohistone H3 (pHisH3) or (d, e, f) PCNA. A single mitotic gure is labeled for pHisH3 immunoreactivity in panel c. Arrows in panels c, e, and f indicate the retinal margin. Abbreviations used: GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. Calibration (50 m) in panel c applies to panels a through c, calibration in panel e applies to panels d and e, and calibration panel f applies only to panel f.

Double-Labeling for BrdU and Markers of Postmitotic Retinal Neurons

We have shown that mitotically active cells at the retinal margin give rise to cells that are integrated into the retina. To determine whether these newly generated cells differentiate into retinal neurons, we doubly labeled retinal sections for BrdU and various neuron-specic markers. One such marker is the RNA-binding protein Hu, which is expressed soon after the onset of differentiation in developing neurons (Marusich et al., 1994). In postnatal chick retina, Hu is expressed by most, if not all, amacrine and ganglion cells (Fig. 4b). At the retinal margin, many amacrine cells were colabeled for BrdU

and Hu immunoreactivity (Figs. 4a 4c). We also probed for BrdU labeling and calretinin immunoreactivity. In mature chick retina calretinin is localized to many amacrine and ganglion cells, a few bipolar cells, and all horizontal cells (Ellis et al., 1991; Fischer et al., 1999). At the retinal margin we detected some amacrine cells that were colabeled for BrdU and calretinin immunoreactivity (Figs. 4d and 4e). Labeling for BrdU and calbindin immunoreactivity also revealed colocalization. Calbindin is expressed by cone photoreceptors, a subset of bipolar cells, a subset of amacrine cells, and a few cells in the GCL (Ellis et al., 1991). At the peripheral margin of the retina, BrdU was localized to the nuclei of calbindin-

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FIG. 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. (c h) Sections of (c e) central and (f k) peripheral retina that have been doubly labeled for (c, f) Pax6 and (d, g) Chx-10. (i k) Sections of peripheral retina that have been doubly labeled for (i) PCNA and (j) Chx-10. Panels e, h, and k are overlaid composites of the two panels immediately to their left. Yellow indicates doubly labeled nuclei. The arrows in panels f through k indicate the retinal margin. Abbreviations used: GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear. Calibration (50 m) in panel e applies to panels a through e, and calibration in panel k applies to panels f through k.

immunoreactive bipolar cells (Figs. 4g 4i); however, we did not detect BrdU in calbindin-positive photoreceptors. This is consistent with the nding that BrdU-positive nuclei were never seen in the ONL. Nevertheless, we found that some cells were colabeled for BrdU and the photoreceptor marker visinin (Figs. 4j 4l) (Yamagata et al., 1990). However, these cells did not have the morphology of mature photoreceptors.

BrdU Labeling of Cells at the Retinal Margin of Form-Deprived Eyes

Form deprivation is known to enlarge the eye and, thereby, cause myopia (reviewed by Wallman, 1993). Excessive eye growth caused by form deprivation is driven by signals derived from the retina that enhance the addition of

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FIG. 4. Sections of chick retinal margin that were double-labeled for BrdU and other markers common to postmitotic retinal neurons. Eyes were exposed to BrdU twice daily from P1 through P5 and enucleated on P14. Sections were doubly labeled for (a, d, g, j) BrdU and (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 retinal 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 composites of the two panels immediately to their left. Abbreviations used: GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear. Calibration (50 m) in panel c applies to panels a through c, and calibration in panel l applies to panels d through l.

proteoglycans to the posterior pole of the sclera (Rada et al., 1994). In control eyes, that received four consecutive daily injections of BrdU, on average, between 5 and 10 labeled cells per section were at the retinal margin (Figs. 5a and 5c). There were more BrdU-labeled cells in the temporal retinal margin than in the nasal retinal margin (Fig. 5c). Form deprivation approximately doubled the number of BrdUlabeled cells at the retinal margin. In treated eyes, between 10 and 25 BrdU-labeled cells were found at the retinal margin (Figs. 5b and 5c). These BrdU-labeled cells were located up to 80 m into the retina, away from the margin. Similar to controls, there were more BrdU-labeled cells in

the temporal margin than in the nasal margin of the retina of form-deprived eyes (Fig. 5c). While visual deprivation increased the number of BrdU-labeled cells that were added to the retina, there was no change in the number of labeled cells that were in the ciliary epithelium adjacent to the retinal margin (Fig. 5c). To corroborate ndings of BrdU-labeling studies, we probed the retinal margin of form-deprived eyes with antibodies to PCNA. After 5 days of visual deprivation, the population of PCNA-immunoreactive cells at the retinal margin was substantially increased compared to that of controls (Figs. 5e and 5d).

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FIG. 5. Effects of visual deprivation on the addition of BrdU-labeled cells to the peripheral margin of the chick retina. Vertical sections of 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 (b) form-deprived from P0 through P5. Calibration 50 m. The histogram in panel c illustrates the number of BrdU-labeled cells in the ciliary epithelium adjacent to the retinal margin and in the INL at the margin of the retina of open or form-deprived eyes. Statistical signicance (*P 0.05; **P 0.005) was assessed by using a paired two-tailed Students t test. Error bars represent the standard deviation of the sample mean (N 6). (d and e) PCNA immunolabeling in vertical sections of the retinal margin obtained from eyes that were (d) left open or (e) form-deprived from P0 through P5. Calibration 50 m.

Effects of Acute Damage on Proliferating Cells at the Retinal Margin

Excitotoxic cell death causes a large increase in the proliferation of progenitors at the retinal margin of the tadpole retina (Reh, 1987). To test whether progenitors at the margin of the postnatal chick retina respond to acute damage with increased proliferation, we injected a single toxic dose of NMDA into the vitreous chamber of newly hatched chicks. NMDA destroys many bipolar and amacrine cells in the central retina of the chick, as indicated by DNA-fragmentation labeling, a symptom of apoptosis (Fischer et al., 1998). In saline-treated retinas, a few nuclei (per section) labeled for DNA fragmentation were detected in the INL within 100 m of the retinal margin (Fig. 6a),

suggesting that some newly generated cells may not survive. Many nuclei in the INL were labeled for fragmented DNA at the peripheral margin of retinas treated with 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 toxintreated retinas (Fig. 6d) compared to the number of labeled cells seen in controls (Fig. 6c). There was, however, an increase in the number of BrdU-labeled cells scattered across the IPL (results not shown). These cells were likely to be activated microglia, consistent with the known location of phagocytes in NMDA-damaged retinas (Fischer et al., 1998). These ndings suggest that the rate of proliferation of progenitors in the marginal zone of the chick retina

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FIG. 6. NMDA-induced damage at the retinal margin does not effect the production of new cells. Vertical sections of retinal margin were labeled for (a, b) fragmented DNA or (c, d) BrdU immunoreactivity. Eyes were injected at P0 with (a, c) saline or (b, d) 2 mol NMDA and harvested at P7 following injection of 0.5 g BrdU at P2 and P3. Arrows indicate the retinal margin. Calibration 50 m.

is uneffected by damage, unlike the progenitors in the marginal zone of the tadpole retina (Reh, 1987).

Effects of Growth Factors on Proliferating Cells at the Retinal Margin

In the goldsh retina, insulin-related growth factors increased the proliferation of progenitors at the retinal margin, while other growth factors had no effect (Boucher and Hitchcock, 1998). Accordingly, we tested whether retinal progenitors at the margin of the avian retina proliferate in response to growth factors. Proliferating cells, labeled with BrdU, were in two distinct populations of cells; those at the retinal margin (Figs. 7a7f) and those in the ciliary epithelium (Fig. 7g). We found that 100 ng per dose of EGF, IGF-I, and insulin induced proliferation of retinal progenitors at the margin, while 10-ng doses or bFGF at either dose did not. Four consecutive daily injections of 10 ng of EGF had no effect upon the number of BrdU-labeled cells in the retinal margin or in the ciliary epithelium (results not shown). Two consecutive daily injections of 100 ng EGF nearly tripled the number of BrdU-labeled cells at the retinal margin (Figs. 7b and 7f). Similar to EGF, 10 ng of bFGF had no effect upon the numbers of cells at the retinal margin that were labeled with BrdU (results not shown). An increased 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-labeled cells in the apical tips of ciliary protrusions (results not shown), indicating that this dose of growth factor was effective, but did not inuence the proliferation of progenitors at the retinal margin. Four consecutive daily applications of 10 ng IGF-I had no effect upon the number of BrdU-labeled cells at the retinal margin or in the adjacent ciliary epithelium (results not shown). However, two consecutive 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 the number of BrdU-labeled cells in the ciliary epithelium and retinal margin (Figs. 7e and 7f). To corroborate these ndings we probed for PCNA immunoreactivity at the retinal margin of eyes injected with growth factors. Consistent with ndings of BrdU labeling, we found that EGF, IGF-I, and insulin, but not bFGF, increased the population of cells at the retinal margin that are immunoreactive for PCNA (Fig. 8). This increase in PCNA immunolabeling was detected at P7, 4 days after the application of growth factors. Assuming that growth factors are cleared from the eye within 24 h after application, these ndings suggest that growth factors have a residual, longlasting effect on proliferation or that PCNA is expressed long after cells have completed their cycle.

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FIG. 7. BrdU labeling at the retinal margin after treatment with various growth factors. Vertical sections of the chick retinal margin were obtained from P7 eyes that were injected on P2 and P3 with 0.5 g BrdU plus (a) saline, (b) 100 ng EGF, (c) 100 ng bFGF, (d) 100 ng IGF-I, or (e) 500 ng insulin. Arrows indicate the retinal margin. Calibration 50 m. The histograms in panels f and g illustrate the effects of saline, EGF, bFGF, IGF-I, and insulin on the number of BrdU-labeled cells in (f) the temporal and nasal retinal margin and (g) the temporal and nasal ciliary epithelium (300 m adjacent to the retinal margin). Signicance of difference (*P 0.05; **P 0.001; ***P 0.0005) between saline- and growth factor-treated eyes was determined by using a two-tailed Students t test. Error bars represent the standard deviation of the sample mean (N 6).

DISCUSSION
Here we provide evidence that a proliferating marginal zone, containing cells that resemble multipotent embryonic progenitors, is present in the retina of postembryonic warm-blooded vertebrates. Our evidence indicates that: (i) proliferation of cells continues into adulthood at the retinal

margin of the chicken eye; (ii) cells at the retinal margin coexpress Pax6, Chx-10, and PCNA and are similar to multipotent retinal progenitors; and (iii) these retinal progenitors produce new neurons that are integrated into the edge of the retina. The proliferating marginal zone of the chick retina is similar to that of sh and amphibians in which a zone of stem cells produces new retinal neurons

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FIG. 8. PCNA labeling at the retinal margin after treatment with various growth factors. Vertical sections of the chick retinal margin were obtained from P7 eyes that were injected on P2 and P3 with 0.5 g BrdU plus (a) saline, (b) 100 ng EGF, (c) 100 ng bFGF, (d) 100 ng IGF-I, or (e) 500 ng insulin. Arrows indicate the retinal margin. Calibration 50 m.

throughout life (Hitchcock and Raymond, 1993; Raymond and Hitchcock, 1997; Hollyeld, 1968; Straznicky and Gaze, 1971; Fernald, 1990). While progenitors in the amphibian retina increase their proliferation and regenerate neurons in response to damage (Reh, 1987), the destruction of cells at the margin of the chick retina does not induce progenitors to increase their rate of proliferation. The nding that the chicken retina contains a proliferating marginal zone came as a surprise to us, not because it contradicts prior reports, but rather because these ndings have gone unnoticed and it has been generally assumed that the retina of hatched chicks does not have a zone of proliferating cells at the peripheral margin. Morris et al. (1976) provided a brief report that [H 3]thymidine accumulates in some cells at the margin of the chick retina during the rst 2 postnatal weeks. Other reports on retinal neurogenesis have focused on the birth dates of different retinal cell types (Fujita and Horii, 1963; Kahn, 1973, 1974; Spence and Robson, 1989; Prada et al., 1991), with only some attention paid to the end of proliferation (Prada et al., 1991). However, the retinal margin has a regenerative capacity in late-stage embryos (Willbold and Layer, 1991), which is consistent with our observations. In central retina, very few BrdU-labeled cells were detected even when BrdU was applied repeatedly, suggesting that these cells are not abundant or that very few of them are dividing. The identity of BrdU-positive cells in the OPL, IPL, and OFL remains uncertain. The shape, size, and

location of these nuclei are characteristic of resident, nonactivated microglia in avian retina (Navascues et al., 1994). Additionally, some of the BrdU-labeled cells in the GCL and OFL could be oligodendroglia. In the chick, myelination of ganglion cell axons in the OFL occurs after hatching (Rager, 1976; Arees, 1978) and may be coincident with glial proliferation, as suggested by BrdU labeling in the GCL and OFL (current study). The generation of these putative 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 retinal cells that were located away from the margin. Furthermore, large numbers of cells were detected at the retinal margin only when BrdU was applied repeatedly. These ndings suggest that progenitors at the retinal margin are dividing and differentiating very slowly and that there is concentric addition of new neurons to the peripheral edge of the retina. The integration of neurons into the retina and acquisition of neural phenotypes occurred much more slowly than during embryonic development. We found that about 80 m of radial length of retina was added in 2 weeks (P0 to P14) of postnatal development, while more than 100 times this length was formed in about 12 days of embryonic

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development (Kahn, 1973, 1974; Prada et al., 1991). Although the character of embryonic and postnatal retinal progenitors is similar, their mitotic activity and rate of neuron production differ greatly. It seems likely that postnatal retinal progenitors do not receive the stimulatory growth factors that enhance their rates of division and production of new neurons. This hypothesis is supported by our ndings that exogenous insulin, IGF-I, and EGF increase the proliferation and addition of new cells to the retinal margin. These ndings indicate that progenitors at the retinal margin are competent to respond to growth factors, but that the concentration of the factors may be very low in the postnatal eye. Alternatively, the preexisting retinal neurons at the margin may secrete inhibitory factors that suppress the activity of these progenitors. While there are many newly generated neurons at the retinal margin, few of these cells were colabeled for visinin immunoreactivity and BrdU. These colabeled cells did not appear to be differentiated photoreceptors, but may have been in an intermediate state of differentiation. In addition, BrdU-labeled nuclei were never found in the ONL, where the nuclei of photoreceptors are located. These ndings suggest that new cone photoreceptors may not be produced at the retinal margin. Since this cell type is formed relatively early during embryonic development (Prada et al., 1991; Spence and Robson, 1989), it is possible that the cues required for the generation of cone photoreceptors are absent in postnatal birds. While BrdU was found in amacrine and bipolar cells, BrdU was not found in horizontal or ganglion cells. BrdU-labeled nuclei were found in the GCL, but these cells did not express Hu. It is possible that these cells might be displaced amacrine cells (that may not express Hu) or a type of ganglion cell that does not express Hu. Taken together, these ndings suggest that amacrine and bipolar cells, but not photoreceptor, horizontal, or ganglion cells, are produced by progenitors at the retinal margin of postnatal chicks. The effects of different growth factors on the proliferation of progenitors at the retinal margin are consistent with the ndings of studies on embryonic retinal progenitors and stem cells in other animals. EGF receptor ligands have been shown to stimulate proliferation of retinal progenitors in rodents (Anchan et al., 1991; Lillien and Cepko, 1992) and promote differentiation of amacrine and Muller cells (An chan and Reh, 1995; Lillien, 1996). It is possible that, in the postnatal chick, exogenous EGF not only induced proliferation of retinal progenitors but also increased numbers of cells that differentiated as amacrine and Muller cells. Con sistent with this hypothesis, we observed that most BrdUlabeled cells in EGF-treated retinas were within the INL, where differentiated amacrine and Muller cells are known to reside. However, in the absence of EGF, BrdU-labeled nuclei accumulate primarily in the INL and, therefore, we cannot say that cell fate was inuenced. Other growth factors used in this study have been shown elsewhere to be mitogenic for retinal progenitors. For example, insulin and IGFs increase the proliferation of stem

Fischer and Reh

cells at the peripheral margin of the goldsh retina (Boucher and Hitchcock, 1998). However, it is puzzling that bFGF was not mitogenic for these cells, since it is a mitogen for progenitors in the developing rat retina (Lillien and Cepko, 1992; Anchan and Reh, 1995). Many studies have demonstrated the importance of FGFs to normal eye development in chick embryos (Park and Hollenberg, 1989; Pittack et al., 1991; Pittack et al., 1997). Thus, there appear to be similarities and differences in types of secreted factors that stimulate proliferation of retinal progenitors in sh, birds, and mammals. However, it is worth noting that most studies of mitogens in sh and mammals have used in vitro assays, while in the present study we have analyzed the effects of these factors in vivo. The addition of new neurons to the margin of the retina may serve to compensate, at least in part, for retinal stretch that is imposed during postnatal ocular growth. When rates of ocular growth were increased by visual 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 proteoglycans to the posterior pole of the sclera (Rada et al., 1994), the retina becomes stretched as the eye grows too much and becomes myopic (Teakle et al., 1993). During normal postnatal growth and excessive growth induced by visual deprivation retinal stretch may induce the progenitors at the margin to increase their rate of division. We propose that mechanical stretch at the retinal margin increased the rates of division of progenitors. In comparison, mechanical stretch increases the proliferation of cardiac broblasts (Booz and Baker, 1995) and cells of the nucleus pulposus (Matsumoto et al., 1999). In sh and amphibians, retinal stretch and the addition of new 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). Although 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 compensate in part for this phenomenon. This annular addition of new neurons to the peripheral margin of the chick retina is similar to the ongoing neurogenerative 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 comments that helped to contribute to the nal form of this paper. The BrdU and Pax6 antibodies developed by Drs. S. J. Kaufman and A. Kawakami, respectively, were obtained from the Developmental Studies Hybridoma Bank developed under auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, Iowa 52242. We gratefully acknowledge the contribution of Myron and Arletta Rasmussen to this study. This

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work was supported by fellowships from the Alberta Heritage Foundation for Medical Research, the Medical Research Council of Canada, and The Fight For Sight Foundation Research Division of Prevent Blindness America to A.J.F. and by an NSF grant (IBN 964843), and the Foundation Fighting Blindness to T.A.R.

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